development of optical trapping for the isolation of ......vetch roots. other rhizobium strains...

University of Reading School of Animal and Microbial Sciences

Development of Optical Trapping for the Isolation of Environmentally Regulated Genes

By

Neil A Schofield

Submitted in partial fulfilment of the requirement for the degree of doctor of philosophy 1998

II

I declare that this is my own account of my research and that this work has not been

submitted for a degree at any other university. I would like to acknowledge the

assistance of members of the laboratory in picking colonies for the final libraries. I

would like to acknowledge that the early vector construction, particularly pPOT, was

carried out jointly with David Allaway.

Neil A. Schofield

III

Abstract

Colonisation of the rhizosphere and nodulation of legumes by Rhizobium is a complex

process involving the expression of environmentally induced genes. The study of these

genes is important in understanding this symbiotic relationship, however the complexity of

the environment makes it impossible to model in the laboratory. In an attempt to identify

these genes, an adaptation of the in vivo expression technology (IVET) strategy using optical

trapping has been used. IVET allows gene expression to be identified in vivo, removing the

need to model the environment.

Using optical trapping, refractive particles, including cells, observed using a microscope can

be trapped and moved using laser light. An optical trap has been constructed that can trap a

single bacteria and isolate it from the population from which it derived. The single cell can

then be grown in liquid culture and studied.

To identify gene expression, a promoter probe vector, pOT1, has been developed. This

vector uses green fluorescent protein as a marker. This allows the observation of promoter

activity by excitation of GFP produced in the cell, using ultra-violet light. A genomic library

of Rhizobium leguminosarum 3841 (LB-2) has been constructed in this vector and a sub

library (LB-3), with no expression of GFP during growth on minimal media, produced.

Exposure of this sub-library to pea roots and the subsequent trapping of the recovered

Rhizobium lead to the identification of rhizosphere induced genes, including thiE. This gene

is important in thiamine biosynthesis.

IVET-OT is an important development in the identification of rhizosphere expressed genes

and its application to other organisms could provide equally important results.

IV

Acknowledgements

I am indebted to my supervisor, Dr Philip Poole, for his guidance and

encouragement. His positive enthusiasm when all I could see was the negative has

kept me going through these long years.

I am particularly grateful to Arthur Hosie, Judith Fry, Emma Lodwig, Liliana

Gilardoni and Mary Leonard without whose help I would still be picking colonies.

My thanks go to all the members of the lab, both past and present, for making it an

enjoyable place to work. In particular I would like to thank David Allaway, for his

continuous support and for having the same taste in music, and Dave Chapman for

keeping me sane during those long nights in the lab. I would also like to thank David

Walshaw and Colm Reid for getting me into this and then leaving.

I would like to express particular thanks to Tony Dawson, who constructed many of

the strange pieces of equipment I designed. His ability to produce finely crafted

apparatus from my scribbled ideas is a wonder.

I am also grateful to everyone in CSS. Their work is indispensable and very much

appreciated. In particular David Butlin who on numerous occasions has rescued me

from the lab, to share some spectacular days in the mountains.

I would like to thank my parents for their love and support, both financial and

mental, through out this project and in the preceding years. Finally, an especial

thanks to my wife, Kath, for her love and prayers, and for putting up with me being

away for so many months.

V

Abbreviations

Ala Alanine

Am ampicillin

AMS acid minimal salts

bp base pair

Cm chloramphenicol

DNA dioxyribonucleic acid

GFP green fluorescent protein

GFPuv UV optimised green fluorescent protein

Gn gentamycin

He Ne helium neon

hr hour

IR Infra red

IVET in vivo expression technology

IVET-OT in vivo expression technology using optical trapping

Kb kilobase

Kn kanamycin

LB Luria-Bertani broth

Met Methionine

min minute

Ne Yag Neodymium – yttrium aluminium garnet

OD optical density

PCR polymerase chain reaction

Phe Phenylalanine

VI

RNA ribonucleic acid

rpm revolutions per minute

Ser Serine

Sp spectinomycin

St streptomycin

Tc tetracycline

Thr Threonine

TT Transcriptional terminator

TY tryptone-yeast media

UV Ultra violet

Val Valine

XP 5-bromo-4-chloro-3-inodolyl phosphate

VII

CONTENTS

CHAPTER 1: INTRODUCTION 1

1.1 Rhizobium 1 1.1.1 Nodulation 1 1.1.2 The nod genes 2 1.1.3 Nitrogen Fixation 4 1.1.4 Nutrient Exchange in the Nodule 5 1.1.5 Carbon Sources in the Rhizosphere 5

1.2 Competition and survival of Rhizobium in the Rhizosphere 6

1.3 Strategies for Identifying Environmentally Induced Genes 10

1.4 Optical Trapping 16 1.4.1 The Characteristics of Lasers 16 1.4.2 Manipulation of particles using Light 18 1.4.3 Forces in Optical Trapping 20 1.4.4 Uses of optical trapping 26

CHAPTER 2: METHODS 30

2.1 Strains 30

2.2 Plasmids 32

2.3 Oligonucleotide primers 35

2.4 Growth conditions 36 2.4.1 Growth media 36 2.4.2 Antibiotics 36 2.4.3 Microtitre plates 37 2.4.4 Stains and fluorescent observations 38

2.5 Molecular Biology 38 2.5.1 DNA Isolation 38

2.5.1.1 Plasmid preps 38 2.5.1.2 Chromosomal preps 39

2.5.2 Restriction digests 40 2.5.3 Ligations 40 2.5.11 Transformation 41 2.5.4 De-phosphorylation of DNA 41 2.5.5 Removal of 5' single stranded DNA 41 2.5.6 Removal of 3’ single stranded DNA 42 2.5.7 Agarose gels and staining 42 2.5.8 DNA Extractions from Agarose Gels 42 2.5.9 Sequencing 42 2.5.10 Conjugations 43 2.5.12 PCR 43 2.5.13 Linker production 45

2.6 Pea growth 45 2.6.1 Surface sterilisation and Germination 45 2.6.2 Pea Growth in Flasks 45 2.6.3 Pea Growth in Universal bottles 47 2.6.4 Nodule harvesting 47 2.6.5 Rhizosphere harvesting 47

VIII

2.7 Optical Trap 48 2.7.1 Microscope 48 2.7.2 Laser 48 2.7.3 Set Up of trap 48 2.7.4 Objective lens 49 2.7.5 Viewing IR laser 50 2.7.6 Microscope Stage 50 2.7.7 Trapping single cells 50

CHAPTER 3: CONSTRUCTION OF AN OPTICAL TRAP 51

3.1 Introduction 51

3.2 Construction of the Trap-Methods and Results 51 3.2.1 Requirements for an Optical Trap 51 3.2.2 Development of the Trap 54 3.2.3 Alignment of the laser 60 3.2.4 Development of the Trapping Protocol 62 3.2.5 Attenuation of the Laser 65

3.3 Isolation and Culturing of Single Cells 69

3.4 Discussion 72

CHAPTER 4: CONSTRUCTION OF PROMOTER PROBE VECTORS 75

4.1 Introduction 75

4.2 Methods and Results 77 4.2.1 Characterisation of reporter genes for use 77 in IVET-OT 77 4.2.2 Construction of pPOT and pGOT vectors 78

4.2.2.1 Cloning of Omega Transcriptional Terminators 78 4.2.2.2 Cloning of phoA reporter gene 79 4.2.2.3 Cloning of antibiotic markers. 82 4.2.2.4 Introduction of Multiple Cloning Sites 84 4.2.2.5 Insertion of GFP reporter gene. 85

4.2.3 Cloning of test promoters in pPOT and pGOT vectors 87 4.2.3.1 Cloning the Lac Z Promoter 87 4.2.3.2 Cloning of the dctA Promoter 87 4.2.3.3 Cloning of the nodC Promoter 88

4.2.4 General fluorescence of pGOT vectors 88 4.2.5 Stability of pGOT vector 89 4.2.6 Construction of pOT1 vector 93

4.3 Discussion 102

CHAPTER 5: CONSTRUCTION OF PROMOTER LIBRARIES 105

5.1 Introduction 105

5.2 Construction of library LB-1: Methods and Results 105 5.2.1 Cloning of 3841 genomic DNA into pOT1 106 5.2.2 The distribution of genomic inserts in LB-1 109

5.3 Construction of the Promoter Library LB-2 112 5.3.1 Construction of LB-2 112

IX

5.4 Construction of Sub-libraries 116 5.4.1 Calibration of plate reader 116

5.4.1.1 The OD630 and OD600 correlate linearly to late logarithmic growth. 117 5.4.1.2 Fluorescence is directly correlated to OD630 118 5.4.1.3 The threshold value for relative fluorescence. 119

5.4.2 Screening of LB-2 using microtitre plate reader 121 5.4.3 Selection of libraries LB-3, LB-4, LB-5 and LB-6 122

5.5 Testing of the library LB-3 123

5.6 Discussion 125

CHAPTER 6: PUTATIVE MEDIA REGULATED PROMOTERS 127


6.2 Screening of library LB-6: Methods and Results 127 6.2.1 Plate Observations 131 6.2.2 Sequence data 131

6.3 Discussion 140

CHAPTER 7 : ISOLATION OF ENVIRONMENTALLY INDUCED PROMOTERS USING IVET-OT 143


7.2 Isolation of LB-3 Clones from the Rhizosphere 144

7.3 Characterisation of the Rhizosphere Isolates. 146

7.4 Discussion 153

CHAPTER8: DISCUSSION 158

REFERENCES 164

1

Chapter 1

Introduction

1.1 Rhizobium

Rhizobium is a small (1-2µm) Gram negative organism found free-living in soil. A

notable part of its lifecycle is its ability to form a symbiotic relationship within the

roots of legumes, culminating in the formation of nitrogen fixing nodules. There are

four genera, within the family Rhizobiaceae, that nodulate legumes: Rhizobium,

Bradyrhizobium, Azorhizobium and Sinorhizobium The work described in this thesis

primarily uses Rhizobium leguminosarum biovar viciae which nodulate pea and

vetch roots. Other Rhizobium strains nodulate different and specific legume roots.

For example, Rhizobium leguminosarum biovar trifolii nodulates clover whilst biovar

phaseoli nodulates bean and R. meliloti nodulates alfalfa.

1.1.1 Nodulation

Nodulation is based on a two-way molecular exchange between the legume and

Rhizobium. The host plant releases signal molecules that stimulate the expression of

genes (nod) within bacteria that are necessary for nodulation. These signal

molecules are flavonoids and have side groups specific to the strain of legume

(Gyorgypal et al., 1991; Peters et al., 1986; Redmond et al., 1986). The nod genes

encode enzymes that are involved in the synthesis of Nod factors, which in turn

cause changes in the morphology of the plant roots. The nod genes and many other

genes required for symbiosis are found on plasmids designated pSym in Rhizobium,

2

Azorhizobium and Sinorhizobium, however, other strains such as Bradyrhizobium

retain them on the chromosome.

The first step in the formation of a root nodule is infection. During infection, the

growth of epidermal hairs on the root surface, are altered resulting in them deforming

or curling (Dazzo & Gardiol, 1984). Bacteria are trapped by this deformation and

begin to invade the root surface. The plant is stimulated to produce a cell wall sheath

known as the infection thread (Callaham & Torrey, 1981). Within the root cortical,

cell division leads to the development of the nodule, where dividing bacteria are

delivered to the cytoplasm of individual plant cell via the infection thread. The

bacteria are engulfed by the plant membrane, which forms a peribacteriod

membrane. Here the bacteria, now termed bacteroids become terminally

differentiated and begin to fix nitrogen within the symbiotic relationship.

Once nodulation is complete, the bacterial cell undergoes the final stages of

differentiation. Cell division and DNA replication cease and the Rhizobium,

surrounded by the peribacteroid membrane, become dedicated to nitrogen fixation.

These terminally differentiated forms are known as bacteroids.

1.1.2 The nod genes

The nod genes fall into two groups, those that are common to all strains and are

necessary for nodulation to occur, and those that are found only in certain strains and

determine host specificity. NodABC comprise the common nod genes, with

mutations in any of them causing a Nod minus phenotype (Debruijn & Downie,

1991). They are found across the range of Rhizobium strains and are functionally

3

interchangeable between strains (Djordjevic et al., 1985; Fisher et al., 1985;

Kondorosi et al., 1984). There are many host specific nod genes for example R.

leguminosarum has nodFECMNTO. Amongst these nodE is particularly important

for the nodulation of peas by biovar viciae or clover by biovar trifolii (Spaink et al.,

1991).

An essential positive transcriptional regulator in the nodulation response of

Rhizobium is NodD. Nod D can occur in single copy or in multiple copies and acts in

a species-specific manner (Honma & Ausubel, 1987; Spaink et al., 1987). The gene

is the only nod gene expressed constitutively. Its N-terminus is highly conserved,

indicating a role in DNA binding. The C-terminus is more variable and it has been

suggested it may have a function such as flavonoid binding (Shearman et al., 1986).

As well as activating the transcription of other nod genes, in R. leguminosarum, it

also regulates its own expression (Rossen et al., 1985).

A conserved region known as the nod box (approximately 50bp) precedes most nod

genes (Rostas et al., 1986). NodD binds this region in two positions and can induce

bending between the two binding sites. How this bending, and the interaction

between NodD and RNA polymerase effect nod gene activation is still a mater of

speculation (Fisher & Long, 1992). The control of expression and the action of

NodD appear to be diverse and complex.

The nod genes encode a series of enzymes necessary for the synthesis of Nod factors.

Individual bacterial strains appear to make a family of Nod factors and substitutions

differ between species (Schultze et al., 1992). The basic structure of a Nod factor is

4

a β-1,4-linked oligomer of N-acetylglucosamine with an N-acyl substitution on the

non-reducing end. The diversity of active Nod factors is reflected in the variation of

nod genes (For a summary of nod genes and function see Fisher et al. table 2 (Fisher

and Long, 1992)).

1.1.3 Nitrogen Fixation

The bacterial genes for nitrogen fixation fall into two broad categories. Those that

have homologies amongst organisms that can fix nitrogen in the free living state are

known as nif. Those that are unique to symbiotic nitrogen fixation are known as fix.

Mutations within these genes result in Rhizobium that are able to nodulate legumes

but are unable to fix nitrogen (Nod+, Fix-)

In R. meliloti, symbiotic regulation of the nif genes appears to be activated by nifA,

whose expression is dependent on low oxygen (Ditta et al., 1987). FixL and fixJ are

part of a two component regulatory system that senses oxygen to activate

transcription in nifA (Davis et al., 1988). A detailed review of this is beyond the

scope of this thesis but has been extensively reviewed by Fischer (Fischer, 1994).

Within the symbiotic nitrogen fixing relationship between plant and bacteria, several

intriguing obstacles arise which need to be overcome by the two organisms. The

nitrogenase enzyme is irreversibly inactivated by oxygen, which is needed for

aerobic metabolism by the bacteroid. However, the function of nitrogenase requires

large amounts of ATP, which the bacterium must provide by aerobic respiration.

These constraints define many of the functions and structures within the symbiosis.

5

Leghemoglobin is an haem-binding protein similar to myoglobin. Its role is to bind

oxygen making it available for respiration while keeping the free oxygen levels low,

thus protecting the nitrogenase (Appleby, 1984).

1.1.4 Nutrient Exchange in the Nodule

The plant supplies carbon derived from photosynthesis to the bacteroids for use in

ATP production. In exchange the plant assimilates ammonia (Long, 1989). It is

generally thought that the carbon supplied by the plant takes the form of C4-

dicarboxylic acids, either L-malate, succinate or fumarate. The Rhizobium

dicarboxylate transport genes (dct) are required for fixation as well as the catabolic

malic enzyme. Mutations in these genes prevent nitrogen fixation, however

mutations in other sugar catabolism genes do not effect fixation (Driscoll & Finan,

1993; Finan et al., 1983). The dicarboxylate transport system is regulated by a two

component sensor-regulator system. Transcription of the gene for dicarboxylate

transport, dctA, is activated by regulation from the product of dctD. The gene dctB

encodes the sensor of dicarboxylate (Reid, 1995; Ronson et al., 1984; Wang et al.,

1989; Watson, 1990).

1.1.5 Carbon Sources in the Rhizosphere

There is a proposed exchange of carbon between the terminally differentiated

bacteroid and free-living members of its strain outside the nodule. Insight into this

came with the discovery of genes in R. meliloti that synthesise a unique compound

known as a rhizopine (Murphy et al., 1987; Murphy et al., 1988). Differentiated

6

bacteroids express the plasmid mos genes, under the control of ntrA-nifA regulation.

The unique rhizopine produced can be catabolised by free living Rhizobium of the

same strain due to set of genes, moc. It had been proposed that rhizopines act as a

exclusive growth substrate, enhancing the competitiveness of free-living members of

the producing strain in the rhizosphere. A recent study, in which moc- and mos-

mutants were compared with wildtype R. meliloti, has indicated that this may not be

the case (Gordon et al., 1996). It appears that the ability to catabolise rhizopines

enhances the rate at which a strain can form nodules when in competition with a

strain unable to catabolise the rhizopine.

1.2 Competition and survival of Rhizobium in the Rhizosphere

The importance of legumes as a crop, combined with nitrogen fixation within the

symbiotic relationship with Rhizobium, has lead to extensive studies of the survival

and competitiveness of Rhizobium. It has been shown that, in controlled conditions,

increased yields of legumes can be achieved by inoculating plants with modified

Rhizobium. However, when these modified strains are inoculated into non-sterile

soil conditions poor results are often obtained for increased yield. This appears to be

due to poor competition of laboratory strains with native strains. Due to the

economic importance of legumes competition has been extensively studied and

reviewed (Dowling & Broughton, 1986; Triplett & Sadowsky, 1992).

For successful nodulation and fixation of nitrogen, the Rhizobium needs to survive,

and to compete with other strains in a series of varied and complex environments.

First, they need to survive and be maintained in the soil. Second, they then need to

7

infect, grow and compete in the rhizosphere. Third, they must compete to initiate

nodulation of the legume.

The factors affecting growth and competition include both genetic and environmental

conditions. The host plant also plays an important role. The environmental factors

are complex, but include the soil type, pH, temperature, water content, salinity, and

competition with microorganisms. An important factor in the ability of a strain to

produce successful nodules is the relative number of that strain surviving in the

rhizosphere. Predators of microorganisms also effect the numbers of Rhizobium, as

do bactericidal agents. For example, the colonisation of Rhizobium is often inhibited

by predation by protozoa (Hossain & Alexander, 1984) as they reduce the numbers

of organisms available for infection. The motility, exopolysaccharide and antibiotic

production by Rhizobium can also effect their competitiveness.

In many cases, the final response is determined by the relationship between these

factors. For example, pH is an important determinant in the ability of Rhizobium to

survive (Glenn & Dilworth, 1994). However, a number of properties are effected by

pH, including motility and chemotaxis. All of these in turn could effect the

competitiveness of an organism, if not its ability to survive. A correlation between

exopolysaccharide production and acid tolerance has been reported (Cunningham &

Munns, 1984). During a pH shift from neutral pH to pH 5.0, no additional proteins

are required. However, within one generation of growth at pH 5.0, an adaptive acid

tolerance response (ATR), with the production of specific proteins that protects the

organism from subsequent acid shock at pH 3.0, occurs (Ohara & Glenn, 1994).

High levels of calcium may also have a significant role in acid tolerance.

8

The response to pH may vary within a strain. R. leguminosarum bv. phaseoli can be

grouped into two symbiotypes, I and II. Type I have a narrow host range whilst type

II have a wider host range as well as being heat, aluminium and acid tolerant. Type

II are more competitive in their nodulation of beans (Phaseolus vulgaris L) in acid

soils, although their numbers and growth do not seem to vary in the rhizosphere

before nodulation (Frey & Blum, 1994).

The natural production of antibiotics by Rhizobium can affect the ability of strains to

nodulate successful. Trifolitoxin (TFX) is a peptide with antibiotic properties,

produced by R. leguminosarum bv. trifolii T24. It inhibits many α-proteobacteria

(Triplett et al., 1994). The effect of this on competition has been established by

cloning the tfx genes into R. etil strain CE3. This strain became more competitive in

rhizosphere colonisation and nodulation (Robleto et al., 1997). A Tn5 mutation of R.

etil strain CE3 designated strain CE3003 has been produced with reduced

competitiveness (Araujo et al., 1994). A fragment containing the open reading frame

rosR compliments the insertion. The gene is homologous to transcriptional

regulators in R. meliloti and Agrobacterium tumefaciens and effects cell surface

hydrophobicity and exopolysaccharide production (Bittinger et al., 1997).

Many genetic markers could effect the competitiveness of Rhizobium. For example,

as means of containing genetically modified Rhizobium released into the

environment, it has been suggested that recA- mutants be used. These were found to

be deficient in their ability for long term survival but not in their short term

persistence (Hagen et al., 1997).

9

Vitamins play an important role in the colonisation of roots by Rhizobium. Growth

of R. meliloti is limited by the availability of biotin, thiamine and riboflavin. These

have been shown to be released from alfalfa roots and promote Rhizobium growth

under laboratory conditions. Biotin is a cofactor binding CO2 in enzymes such as

pyruvate carboxylase in R. etli (Dunn et al., 1996). The requirement for biotin could

therefore be linked to the CO2 requirement of Rhizobium and Bradyrhizobium for

growth. It has been shown that both uptake and biosynthesis of biotin contribute to

successful root colonisation (Streit et al., 1996). Rhizobium responds to plant-

derived biotin and a biotin uptake system or genes responding to biotin can be

postulated to exist. A DNA locus, bioS, has been identified that responds to external

biotin. Although cells with mutations in this locus accumulated biotin faster, they

were less competitive on low biotin laboratory media while showing no difference in

competition with wild type in the rhizosphere (Streit & Phillips, 1997). The

identification of the importance of vitamins in the symbiosis of Rhizobium has been

identified, however, the processes involved are still being characterised.

The response of Rhizobium to its environment and infection of legume roots is

probably one of the best-studied plant/bacterium symbiotic relationships. However,

the complexity of this association means that it is still poorly understood. While

determining the factors that govern competition and survival in the environment is a

complex problem, it is a key aim in environmental microbiology. A first step would

be to determine which genes are specifically switched on in the rhizosphere. These

genes must code for functions affecting growth and competition. To identify such

genes we developed a genetic system that can identify rhizosphere expressed genes

10

whilst giving spatial information about this expression. We have designated this as

in vivo expression technology using optical trapping IVET-OT. Below the original

IVET strategy, developed for studying pathogenicity in bacteria, is described.

1.3 Strategies for Identifying Environmentally Induced Genes

In recent years, the study of the genetics of the interactions between pathogens and

their hosts has developed. Physiological studies on these interactions are often

difficult due to the complexity of the relationships. For this reason, genetic and

molecular techniques are routinely used in their study.

A wide number of approaches are available for determining the genetics of these

interactions. In general the aim of determining the nature of pathogen-host

relationship has been the search for methods of controlling disease by reducing the

pathogenicity and virulence of organisms.

Numerous techniques have been developed to study the differential expression of

genes. Studying the effect of mutations can give an important insight into the

function of a gene. Mutations can be direct or random. In direct mutagenesis a

cellular function is deemed to be important. Disrupting or deleting the gene develops

a mutant strain, which is then compared with the wild type strain. This technique is

limited to identify genes with a postulated function. Random mutagenesis can be

carried out using UV or chemicals, with no postulation of a gene's function.

Mutations in a bacterial trait can be identified and the genes involved cloned.

Problems can arise with this method due to multiple mutations.

11

The use of transposons to cause random mutations can overcome the problem of

multiple mutations and results in the mutation being tagged. This has been greatly

enhanced by the development of small mini-transposons. The use of many of these

genetic techniques has been reviewed by Hensel (Hensel & Holden, 1996).

Gene fusion techniques, either transcriptional or translational, have been developed.

For example, random fragments of the bacterial genome are inserted upstream of a

promoterless reporter such as lacZ. Changes in gene expression in response can then

be monitored directly.

An alternative approach is to study gene expression by studying variations in RNA

expression. During a particular process it can be assumed that genes necessary for

that function, for example root colonisation, are transcribed only at that time. The

synthesis of cDNA from RNA allows the identification of genes transcribed during

infection. Subtractive hybridisation is often used where cDNA from an uninduced

condition is hybridised with cDNA from a test condition. The unhybridised cDNA

remaining is specifically expressed in the test environment.

An interesting development of the study of RNA and differential display is the use of

arbitrarily primed PCR (McClelland et al., 1995). When carrying out PCR on

complex templates, the primers used can be either random, where each base is

represented at each position, or arbitrary, where there is only a single base at each

position on the primer. Arbitrary primers are sequence dependent but are not chosen

to represent specific template binding sites. A good match between primer and

12

template may therefore only consist of 6-8 bases in the 10 bases at the 3' end or the

primer. With these primers, after the first round of amplification, the single stranded

PCR product has the primers at each end and can be further amplified. This results

in the most prominent PCR products resulting from the most successful primer pairs.

In RNA fingerprinting, the first stage involves reverse transcription. With eukaryotic

RNA this can be carried out using the primer based on the oligonucleotide (dT), to

prime at the poly-adenylated tail, followed by two bases at the 5' end. Since bacterial

RNA is not poly-adenylated an arbitrary primer must be used. This allows sampling

anywhere in the RNA including open reading frames. The second stage of the

procedure involves PCR with arbitrary primers.

The PCR fingerprints obtained using these methods can be compared. The profiles

between different conditions under which the organism is grown can be compared.

This has the advantage that a large number of overlapping conditions can be

compared simultaneously, allowing genes expressed under a number of conditions to

be identified. However one draw back is that rare RNAs may be underrepresented as

the probability of observing a product is a function of both the best primer match and

the abundance of the RNA. This can be overcome by using nested priming

fingerprints. In this case the original fingerprint is reamplified with an arbitrary

primer based on the original but with the bases shifted 3' with additional arbitrary

primers. These RNA fingerprinting techniques are reviewed by McClelland et al.

(McClelland, et al., 1995).

13

Messenger RNA levels can also be analysed by hybridisation to DNA dot blots

(Chuang et al., 1993). In this technique an overlapping set of clones in lambda,

spanning the genome of E. coli was constructed. Induced or repressed genes in each

cloned region are identified using hybridisation to DNA dot blots. RNA is extracted

from the cell and reverse transcription carried out with 32P labelled dCTP and a

random primer. The total cDNA is then hybridised to a library of DNA dot blots.

Comparison of the clone under control and experimental conditions indicates the

differential expression of genes.

The complexity of the molecular communication between host and pathogen makes

modelling it in the laboratory difficult, leading to limitations in many of the

approaches above. It has, for example, been very difficult to determine exactly

which genes need to be expressed by a pathogen to establish successful infection of a

host. Recent approaches that overcome the need to model the host stimulation, by

using a fully functional host, include signature tagged mutagenesis (STM) and in

vivo expression technology (IVET). Both these approaches were developed for

Salmonella typhimurium.

STM is a development of other mutagenesis techniques, overcoming one of their

major drawbacks by allowing a mutant with attenuated virulence to be identified

within a large population of mutants. In this technique, each transposon mutant is

marked with a DNA tag consisting of a 40bp variable region surrounded by a

constant region allowing PCR amplification of the tag. The mutants are plated in

microtitre plates from which DNA colony blots are prepared. An inoculum of the

pooled mutants is used on the host mouse from which a pool of recovered mutants is

14

obtained after infection. The tags from the inoculum pool and the recovered pool are

amplified and used as a probe on the DNA blots. Mutations in virulence genes, are

absent from the recovery pool as they are not virulent and do not survive in the host.

Mutations are obtained, that hybridise with the inoculum pool but not the recovery

pool, that are therefore attenuated in virulence (Hensel et al., 1995). Using this

method, a new pathogenicity region in S. typhimurium has been identified (Shea et

al., 1996). It has been noted that some mutations may be missed if another mutation

produces a virulence function that allows the mutant to grow in the host. This

system also suffers from the limitations of insertion mutagenesis. The transposon

may still have effects on genes further downstream of its insertion point. There is

also the problem of non-random insertion of transposons.

The IVET strategy is a development of the use of transcriptional and translational

fusions. In the original method, a promoter fusion was made with a promoterless

purA and LacZY gene fusion. This was inserted into the chromosome of a purA

auxotroph of S. typhimurium using the suicide vector pIVET1 based on pGP704.

Recombination between the inserts in the vector and the homologous region in the

chromosome occur to produce a tandem insert with the native gene and the reporter

gene regulated by the native promoter. In this way full functionality is maintained in

the cell. Selection for ampicillin resistance drives integration of the recombinant

plasmid into the chromosome. During infection of a mouse, only active fusions

resulting in the complementation of pur survived. These were then recovered from

the mouse spleen and screened for Lac- in vitro. Of the cells recovered from the

mouse, 5% had sufficient purA transcription to survive within the mouse but were

15

Lac- on laboratory media. Novel genes important in virulence were isolated from

this group (Mahan et al., 1993).

One of the limitations of this version of IVET is that it relies on complementation of

an auxotroph. If a gene is only transiently expressed, insufficient transcription may

occur resulting in important factors being missed. Several adaptations of IVET have

been developed that overcome this (Camilli & Mekalanos, 1995; Mahan et al.,

1995). The technique has been successful in identifying many new genes involved in

virulence. For example 11 strains of Staphylococcus aureus have been isolated

containing previously unknown virulence genes (Lowe et al., 1998).

In our adaptation of the IVET strategy, know as IVET-OT, we use a plasmid-based

system for ease of manipulation. We also do not use auxotrophs thereby eliminating

the problems of transiently expressed genes not leading to survival of an auxotrophic

host. The use of fluorescence technology and green fluorescent protein (GFP) has

allowed visualisation of the active promoters and allows transient expression to be

monitored as GFP is stable for long periods. In the study of the infection of plant

roots by Rhizobium, spatial information about the expression of genes is of

considerable value and can be visualised using GFP (Gage et al., 1996). The

physical process of nodulation has been studied using microscopy and it was our aim

to combine this with a genetic study. Combining IVET using a fluorescent marker

with the micromanipulation achievable with optical trapping has given us a powerful

tool in the study of the legume-Rhizobium symbiosis that could be adapted to many

other host-pathogen relationships.

16

In this strategy, a plasmid based promoter library is constructed in Rhizobium. The

constitutive promoter fusions are removed to produce a silent promoter library. This

sub-library is inoculated into the rhizosphere and allowed to grow. Individual cells

that express a reporter under these conditions are recovered using a light based

micromanipulator known as an optical trap. The isolated cell is grown up and

analysed.

1.4 Optical Trapping

Microscopy is a powerful tool for observing and identifying biological specimens.

However, light is versatile and can be used for far more than the simple observation

of microscopic organisms. Light is the basis of microscopy and spectroscopy and the

use of fluorescent and phosphorescent probes have allowed the measurement of a

wide range of cellular processes. Light based systems are also being used to replace

radioisotopes. The properties of laser (Light Amplification by Stimulated Emission

of Radiation) light are being utilised in a wide range of tools, including confocal

microscopes, gene sequencers and cell sorters.

1.4.1 The Characteristics of Lasers

Lasers use stimulated emission to produce a light beam with specific properties.

This occurs when electromagnetic radiation causes an atom in a high-energy state to

emit radiation and fall to a lower energy state. The emitted radiation adds to the

incident radiation causing an amplification effect. There are four main types of laser;

gas lasers, solid state lasers, dye lasers and semiconductor lasers. These produce

light beams with similar general properties. The beam has a high intensity and

17

combining this with the collimation of the light, it can be projected over a large

distance as a defined beam. The coherence of the light allows high contrast

interference fringes to be formed. This is important in imaging and focusing of the

laser. Lasers also have spectral purity that has allowed them to be used in high-

resolution spectroscopy.

Another important property of lasers utilised in optical trapping is the precise cross

sectional profile of the beam. For optical trapping the beam needs a Gaussian

distribution of light intensity across the beam. For a laser oscillation to occur the

mirrors at each end of the laser cavity must be positioned to form a resonant cavity.

A wave within the cavity will then replicate itself so that the electrical fields add in

phase. The axial modes produced in this way are discrete narrow spectral lines and

form the basis of the fluorescence line width for the laser output. These modes are

formed by plane waves travelling exactly along the axis joining the centre of the

mirrors and contribute to a single spot of light in the laser output. Within the cavity

however there are waves travelling just off axis. These replicate along a more

complex path and give rise to transverse electromagnetic or TEM modes. These

modes are characterised by a pattern of spots if the laser is shone on a screen. The

minima in the horizontal plane is given by the term q whilst those in the vertical

plane are given as r. Thus the modes can be described as TEMqr (Fig1.1). For

optical trapping a TEMoo laser is generally used (Hecht, 1992; Wilson & Hawkes,

1987).

18

TEM00 TEM01 TEM02

TEM10 TEM11 TEM12

TEM20 TEM21 TEM01*

1.4.2 Manipulation of particles using Light

A more unusual property of light is its ability to produce radiation pressure. When

dielectric material refracts light, tiny forces are produced. Using these forces a

microscope becomes a tool for manipulating biological material not just for

observing it. Mechanical tools such as glass capillaries and needles have been used

for manipulations however these have several disadvantages. They are limited in

their use as they can cause damage to delicate biological material. Sterility is

difficult to maintain and they are limited in their use by the size that tools can be

Fig. 1.1. The patterns of some of the possible transverse electromagnetic modes (TEM) achievable with lasers are shown. TEMqr gives the number of minima in the horizontal (q) and vertical (r) plane. TEM01* represents the donut like pattern achieved by combining TEM01 with TEM10 profiles, giving a central minima.

19

produced. Light on the other hand overcomes these problems. Sterility is easily

maintained as no mechanical parts are introduced to the sample. The limit on the

size of the material to be manipulated is on the scale of the wavelength of the light.

This opens up the possibility of manipulating bacteria and viruses. Damage to

biological material can be limited as the pressures are smaller on any one part of the

cell and the forces associated with the light can be limited to a volume a few

wavelengths across. This also introduces one of the great advantages of light as a

manipulator, namely the ability to work within a cell without opening the cell first.

This is most notably seen when using Laser microbeams to cut or perforate an

organelle within a cell. A High numeric aperture 100X objective lens focuses the

light under a high angle. This means the power density just 3µm from the focus is an

order of magnitude smaller than at the focus. In practical terms this means that if

work is carried out a factor of 2 above the threshold for an effect, the effect is no

longer produced 1-2µm from the focus. This allows an organelle to be cut with a

microbeam without damaging the cell wall or, in the case of the optical trap, a cell to

be trapped without affecting or trapping its neighbours (Greulich & Weber, 1992).

The use of light to manipulate bacteria cells and viral particles using an argon laser

trap was first demonstrated by Ashkin et al.(Ashkin & Dziedzic, 1987). Later optical

traps used less damaging infrared (IR) lasers to trap and move both free bacteria and

particles within the cytoplasm of spirogyra (Ashkin & Dziedzic, 1989; Ashkin et al.,

1987). Since then numerous applications have been suggested and tried (Ashkin,

1980; Block, 1992; Chu, 1991) including the isolation and culturing of single yeast

(Grimbergen et al., 1993) and bacteria (Mitchell et al., 1993).

20

1.4.3 Forces in Optical Trapping

Optical trapping utilises the property of coherent light to produce tiny forces when

refracted. These forces, known as radiational pressure, are very small; several

milliwatts of laser power giving only a few piconewtons of force. This is however

sufficient to pull a bacterium 10 times faster than it can swim (Block, 1992).

For trapping to occur, a complex balance of forces is necessary. The net force is a

balance between refractive or gradient forces and radiational pressure due to

reflection and absorption. Radiometer forces due to thermal gradients are also

produced (Afzal & Treacy, 1992)

For particles larger than the wavelength of the light, a simple ray optics model using

radiation pressure is sufficient to explain the forces needed for trapping. Forces due

to refraction occur because of momentum changes in the photons within the light

beam. Before entering the cell the parallel beams of light can be thought of as

having primarily vertical momentum. After refraction however the light has picked

up horizontal momentum. Due to conservation of momentum, an equal and opposite

force is conveyed to the cell (Fig.1.2) (Block, 1992).

21

The Radiation pressure is dependent on the refractive indices of the particle and its

surrounding medium. Fig 1.3 shows the optical forces on a particle larger than the

wavelength of the light. In a, the refractive indices of the particle (n1) is greater than

the medium (n2). Refraction of the light ray (a) by the particle causes a momentum

change ∆P=P1-P0 where P1 is the momentum after refraction and P0 is the momentum

before refraction (see insert Fig 1.3). Due to the laws of conservation, a force, -∆P is

conveyed on the particle. This is also true for ray (b) and the sum of -∆P for all the

rays results in an overall radiation force, F, in the direction of the focal point, f.

Path of photon

Cell membrane

Refraction of photon by membrane

Resultant force on cell

Change in momentum

Fig. 1.2. A photon hitting the cell wall is refracted. Due to conservation of momentum, the change in momentum causes a momentum change on the membrane.

22

-∆P

Fig. 1.3. Ray optics diagram showing the effect of two rays, a and b striking a particle when the refractive index of the particle (n1) is a) greater than the refractive index of the medium (n2), b) less than the refractive index of the medium and c) when the particle is reflective. f is the focal point of the incident laser beam. The radiation force (F) is the sum of the change in momentum (-∆P) induced by the refraction of the light rays (Insert) [Sasaki, 1992 #71].

a

a

a

b

b

b

P1

P0

∆P

-∆P

-∆P

-∆P-∆P

-∆P

-∆P

-∆P

-∆P

-∆P

f

F

f F

f

a) n1>n2

c) Particle highly reflective

b) n1<n2

23

If n1 is less than n2 then the situation is reversed and the overall force is opposite to

that of the laser (Fig 1.3b). In this case the particle is repelled from the focal point.

If the particle is reflective, simple ray optics indicate that it will be repelled in a

similar fashion to particles with n1<n2 (Fig 1.3c)(Sasaki et al., 1992). To be able to

trap particles repulsed from the laser, Sasaki et al. developed a trap using a scanning

laser of TEM00 type. This configuration causes a "well" of low intensity light on the

axis of rotation, into which a particle is repelled from the spinning high intensity area

around it. A similar effect can be achieved using TEM*01 lasers. An alternative

method for trapping particles with low refractive index, proposed for using optical

traps in micromechanics, is to fabricate the object to be trapped into a donut shape.

In this way, it has been shown that a non-scanning TEM00 laser can trap the object by

exerting force on the inner wall (Higurashi et al., 1995).

For particles less than the wavelength of the light, particularly dielectric particles

such as cells, models where light is described as an electromagnetic field need to be

considered. The basic process involves the induction of dielectric dipoles within the

object to be trapped. The electromagnetic field causes a charge separation within the

object and attracting forces are generated between the field and object. These forces

were first used in trapping and cooling atoms, where the particle achieves a lower

energy state by moving into the focal point of the Laser (Chu, 1991).

24

Intensity of laser light

Cross-section of laser A B

A B

Laser prior to focusing by objective

Fig. 1.4. The cross section of a TEM00 laser has a Gaussian distribution.

If the energy intensity within the incoming beam were uniform the forces on the cell

would balance out resulting in no net movement. However the intensity within a

TEM00 beam is greatest at its centre (Gaussian distribution) creating a gradient

(Fig.1.4). This causes an imbalance in the forces on the cell resulting in a net

movement towards the brightest point of the gradient where the photons have

greatest energy (Block, 1992).

Rays of light hit the cell across its whole diameter, however only those further from

its centre cause relevant trapping forces (Fig 1.5). Light passing through the centre

of the cell cause forces due to the change of momentum at the two membrane

boundaries that are equal and opposite. These forces therefore cancel out. Rays'

25

incident nearer the edges of the cell are refracted in such a way as to give momentum

changes with a resultant backward component (Afzal and Treacy, 1992). It can

therefore be seen that within a cone of rays, those near the axis contribute little to

trapping whereas those nearer the periphery give the greatest trapping forces.

a)

c)b)

iii ii

i

ii ii i i

Fig. 1.5. Within the focal cone of the laser (a), beams of light (i)that pass through the centre of the cone do not contribute to the trapping forces as the forces created (F) cancel out (b). Those that are acutely focused (ii) contribute most significantly to the trapping forces (c).

F

F FF F

FF

FF

26

This is further confirmed by work done by Sako et al. (Sato et al., 1991). They used

a TEM*01 laser to achieve trapping. This beam has a doughnut like profile with the

highest intensity in a disk around the outside. It was found that a TEM*01 laser gave

20% stronger horizontal force in the trap than a TEM00 laser at the same power.

To achieve gradient forces large enough to balance the radiational forces, the cone

angle must be large. This is achieved by using a high numeric aperture (NA) lens.

Afzal et al. found that a lens with NA 0.85 gave a 40° cone angle whilst a lens with

NA 1.25 gave a 70° cone angle (Afzal and Treacy, 1992). The NA 0.85 was

sufficient to trap polystyrene beads or 2 and 3µm but for 1µm beads the NA 1.25 was

necessary.

The stability of optical traps has now been predicted using computer models (Bakker

Schut et al., 1991).

1.4.4 Uses of optical trapping

The ability to use light to manipulate material at a microscopic level, and to

manipulate internal structures within a cell without damaging the cell membrane

(Ashkin and Dziedzic, 1989) has lead to a wide range of applications being

developed. Single beam optical traps have been incorporated into a confocal

microscope to extend its capabilities (Visscher & Brakenhoff, 1991). It is possible to

accurately calibrate the forces involved in trapping (Sato et al., 1991) and this has

lead to the technique being used to measure internal cellular forces. The forces

27

needed to drive organelles along microtubules in the amoeba Reticulomyxa were

calculated to be 2.6x10-7 dynes for a single motor molecule. This was calculated by

trapping mitochondria and measuring the trapping force at the point the mitochondria

escapes the trap (Ashkin et al., 1990). Optical trapping protocols have been

developed for the study of kinesin molecules interacting with microtubules (Block et

al., 1990) and have been used to measure the forces involved in the interaction (Kuo

& Sheetz, 1993). Actin and myosin interactions have also been measured using

optical trapping (Finer et al., 1994; Spudich, 1994). In this case two optical traps

were focused on beads attached to a single actin filament. A microscope slid was

coated with skeletal muscle heavy meromyosin (HMM). Polystyrene beads coated in

HMM were applied to the actin filament, which was brought close to the slide. The

displacement and force on the filament stretched between the two traps could then be

studied. Optical traps have also been used to measure the unbinding and rebinding

forces of actin and myosin molecules (Nishizaka et al., 1995). Another important

molecular motor, driving flagella, has also being studied using optical trapping

(Block et al., 1989; Block et al., 1991).

Another target molecule for study using optical trapping is DNA. Microsurgery

using laser based optical scalpels has been carried out on chromosomes (Seeger et

al., 1991). The use of optical traps has allowed specific interactions to be produced

between dissected fragments, for example dissected chromatids during metaphase

could be kept together until anaphase (Liang et al., 1993). This ability to combine

optical traps with optical scalpels to produce non-contact manipulation techniques

that can be used in sterile environmentally controlled chambers could be used to

supersede established techniques. For example, the study of fertilisation and embryo

28

development by trapping gametes could be carried out (Schütze & Clement-

Sengewald, 1994).

Another form of microsurgery using optical traps was demonstrated by Steubing et

al. (Steubing et al., 1991). Highly selective cell fusion was carried out by placing

two cells (myeloma cell line NS-1) together using an optical trap. The cells were

fused to form a viable hybrid cell using pulses from an UV laser microbeam. The

pinching and local binding of membranes has been studied and a theoretical model

proposed (Bar-Ziv et al., 1995). Bar-Ziv et al. have also studied the effect that

trapping has on the tension in lipid vesicles (Bar-Ziv et al., 1995). They observed

that tension was induced in membranes by lipid material being attracting into the

trap. Fluctuations in the vesicles are suppressed causing them to become pressurised

and when the trap is shut off, to expel inner vesicles.

The ability to bring cells together has also been used to study of the interactions

between cells in the immune response. For example natural killer cells and human

erythroleukemia cells have been forced together using an optical trap, resulting in the

blebbing of the target cell membrane (Seeger, et al., 1991). Further work of this type

could increase understanding of the immune response.

An interesting use of optical trapping was its use in the induction of cell wall growth

in the green alga Chara vulgaris (Leitz et al., 1995). This alga senses gravity using

rhizoids. If the statoliths are positioned at one end of a horizontal rhizoid, simulating

the effect of gravity, differential growth of the opposite cell wall was observed.

29

The ability to isolate single cells from a mixed population of cells has been utilised in

the isolation of a hyperthermophilic archaeum from an environmental sample (A hot

pool in Yellowstone Park, Wyoming). The strain, which could not be isolated by

classical enrichment and plating techniques, was identified by 16S rRNA sequence to

be part of the population. A fluorescent oligonucleotide probe identified the cell

morphology and a cell with that morphology was trapped, isolated and grown (Huber

et al., 1995).

As can be seen from this summary, optical trapping is a powerful and versatile tool.

Its uses are widespread and it is set to become a major tool in the biologists arsenal.

In this project we apply optical trapping to the problem of gene expression by

combining it with IVET. It is used to isolate individual clones expressing a marker

from a complex environment without the need for intermediate growth. This

technique has been designated IVET-OT.

30

Chapter 2

Methods

2.1 Strains

Strain Description Reference/source

DH5α E. coli: supE44 DlacU169 (f80 lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1

(Hanahan, 1983)

R. leguminosarum 3841

Spontaneous streptomycin resistant derivative of R. leguminosarum bv. viciae strain 300

(Johnston & Beringer, 1975)

RU1080 Conjugation of pGOT-K into 3841 This work RU1081 Conjugation of pGOT-K-lacZ into 3841 This work RU1087 Conjugation of pGOT-K-dct into 3841 This work RU1088 Conjugation of pGOT-K-nodC into 3841 This work RU1158 pOT1 library controls, high expression of GFP This work RU1159 pOT1 library controls, low expression of GFP This work RU1160 pOT1 library controls, no expression of GFP This work RU1182 LB-6 library isolate (pOT1) This work RU1183 LB-6 library isolate (pOT1) Insert DNA

homologous to fixND This work

RU1184 LB-6 library isolate (pOT1) Insert DNA homologous to 16S rRNA dimethyltransferase

This work

RU1188 LB-6 library isolate (pOT1) Insert DNA homologous to metX. Expression increased in late stationary phase.

This work

RU1197 LB-6 library isolate (pOT1) This work RU1198 LB-6 library isolate (pOT1) This work RU1199 LB-6 library isolate (pOT1) This work RU1201 LB-6 library isolate (pOT1) This work RU1202 LB-6 library isolate (pOT1) This work RU1203 LB-6 library isolate (pOT1) This work RU1204 LB-6 library isolate (pOT1) This work RU1207 LB-6 library isolate (pOT1) This work RU1209 LB-6 library isolate (pOT1) This work RU1212 LB-6 library isolate (pOT1) This work RU1214 LB-6 library isolate (pOT1) This work RU1215 LB-6 library isolate (pOT1) This work RU1216 LB-6 library isolate (pOT1) Insert DNA

homologous to Phytochrome This work

RU1220 LB-6 library isolate (pOT1) This work RU1226 LB-6 library isolate (pOT1) This work RU1230 LB-6 library isolate (pOT1) This work RU1234 LB-3 library isolate induced by phosphate

limitation This work

31

Strain Description Reference/sourceRU1235 LB-3 library isolate induced by phosphate


RU1236 LB-3 library isolate induced by Succinate and hesperitin

This work

RU1242 LB-3 library isolate induced by phosphate limitation

This work


This work


This work

RU1245 LB-3 library isolate induced by hesperitin This work RU1246 LB-3 library isolate induced by phosphate



This work


This work


This work


This work


This work

RU1253 LB-6 library isolate (pOT1) This work RU1256 LB-6 library isolate (pOT1). Expression

increased in late stationary phase. This work

RU1257 LB-6 library isolate (pOT1) This work RU1302 LB-3 library isolate. Insert DNA homologous

to thiE and thiM This work

RU1311 pOT1 library standard 2000 fluorescence units This work RU1312 pOT1 library standard 10,000 fluorescence

units This work

RU1313 pOT1 library standard 20,000 fluorescence units

This work

RU1314 pOT1 library standard 30,000 fluorescence unit This work RU1315 pOT1 library standard 40,000 fluorescence

units This work


This work


This work


This work


This work

32

2.2 Plasmids

Plasmid Host strain

Details Reference/source

pBB-Gm-Gfpuv DH5α Intermediate in promoter probe construction: GFPuv NcoI/StuI reporter cartridge including TT cloned into pBBR1-MCS-5

This work

pBBR1MCS DH5α Broad host range cloning vector. CmR

(Kovach et al., 1994)

pBBR-MCS-5 DH5α Broad host range cloning vector. GnR

(Kovach et al., 1995)

pGEM-T DH5α Cloning vector for PCR products. Supplied pre digested with 3’ thymidine. AmR

Promega Ltd.

pGFP DH5α Source vector for gfp. pUC19 ori AmR

Clontech laboratories inc.

pGFPuv DH5α Source vector for gfpuv. pUC19 ori AmR

Clontech laboratories inc.

pGOT-k DH5α Promoter probe vector. pBBR1MCS replicon with promoterless gfp reporter. knR

This work

pGOT-k-dct DH5α dctA promoter cloned into pGOT-k. knR

This work

pGOT-k-lacZ DH5α LacZ promoter cloned into pGOT-k. knR

This work

pGOT-k-nodC DH5α nodC promoter cloned into pGOT-k. knR

This work

pGOT-S DH5α Promoter probe vector. pBBR1MCS replicon with promoterless gfp reporter. spR

This work

pHP45 Ω DH5α Omega interposon stR spR (Prentki & Krisch, 1984) pIJ1814 ET12567 pUC18 derivative containing nod

box and nodC. AmR A.Downie

pJQ254 DH5α Cloning plasmid with NotI–SmaI-NotI in MCS. KnR

(Quandt & Hynes, 1993)

pMP220 IncP transcriptional fusion vector. TcR

(Spaink, et al., 1987)

pND-1 DH5α Intermediate in promoter probe construction: TT and phoA cloned into pSK as SacII/NotI fragment

This work

pND-2 DH5α Intermediate in promoter probe construction: TT and KnR cloned into pND-1 upstream of phoA

This work

pND-4 DH5α Intermediate in promoter probe construction: gfpuv and TT (Pharmacia) cloned into pOTK as SpeI cartridge. knR

This work

33

Plasmid Host strain


pNEB193 PUC19 derivative with unique cloning sites: AscI, PacI, PmeI. AmR

New England Biolabs

inc.

pOT1 DH5α Promoter probe vector based on pBBR-1-MCS-5 replicon. Contains promoterless gfpuv and MCS between two unique transcriptional terminators. GnR

This work

pOTK DH5α pBBR1MCS replicon with phoA reporter between two TTs. knR

This work

pOTS DH5α pBBR1MCS replicon with phoA reporter between two TTs. SpR

This work

pPOT-k DH5α Promoter probe vector. pBBR1MCS replicon with promoterless phoA reporter. knR

This work

pPOT-S DH5α Promoter probe vector. pBBR1MCS replicon with promoterless phoA reporter. StR

This work

pRAT1 DH5α Plasmid containing KnR cartridge D. Evans

pRK2013 ColE1 replicon with RK2 tra genes. Used for mobilising incP and incQ plasmids. KnR

(Figurski & Helinski,

1979)

pRU160 DH5α Omega transcriptional terminator from pHP45 Ω cloned into pGEMT

This work

pRU161 DH5α Omega transcriptional terminator from pHP45 Ω cloned into pGEMT

This work

pRU162 DH5α SacRB PCR fragment cloned into pGEM-T

This work

pRU279 DH5α Intermediate in promoter probe construction: knR BamHI fragment from pRAT1 cloned into pRU160

This work

pRU496 RU1182 pOT1 derivative isolated from RU1182. GnR

This work

pRU498 RU1188 pOT1 derivative isolated from RU1188. Insert DNA homologous to metX. Expression increased in late stationary phase. . GnR

This work

pRU499 RU1183 pOT1 derivative isolated from RU1183. Insert DNA homologous to fixND. GnR

This work

pRU500 RU1184 pOT1 derivative isolated from RU1184. Insert DNA homologous to 16S rRNA dimethyltransferase. GnR

This work

34

Plasmid Host strain


pRU504 RU1302 pOT1 derivative isolated from RU1302Insert DNA homologous to thiE and thiM. GnR

This work

pRU509 RU1197 pOT1 derivative isolated from

RU1197. GnR This work


This work


This work


This work


This work


This work


This work


This work


This work


This work


This work


This work


This work


This work

pRU529 RU1216 pOT1 derivative isolated from RU1216. Insert DNA homologous to Phytochrome. GnR

This work


This work


This work

pRU534 RU1256 pOT1 derivative isolated from RU1256. Expression increased in late stationary phase. GnR

This work


This work


This work

pRU569 DH5α Inset from pRU504 cloned into pNEB193 as pacI fragment

This work

35

Plasmid Host

strain Details Reference/source

pRU61 DH5α 0.9Kb dcta-B in BluescriptII SK-. AmR

(Reid & Poole, 1998)

BluescripII pSK-

DH5α Phagimid, pUC19 derivitive, f1(-) origin of replication, ColE1 replicon. AmR

Stratagene Ltd

BluescripII pSK+

DH5α Phagimid, pUC19 derivitive, f1(+) origin of replication, ColE1 replicon. AmR

Stratagene Ltd.

pTR101 TG1 Broad host range cloning vector containing ParDE. RK2 derivative. AmR tcR

(Roberts et al., 1990)

pWS233 HB101 pSUP101 derivative containing sacRB

A. Puhler

2.3 Oligonucleotide primers

NAME SEQUENCE 5' to 3' DESCRIPTION USE Supplier P12 GGCTTTACTAAGCTGATC

CGGTGG 5' end of omega transcriptional terminator

PCR Genosys

P13 GGGGATCCGGTGATTGATTGAGCA

3' end of omega transcriptional terminator

PCR Genosys

P19 CCACCCGACAGCGAAAATTCAC

C terminus of phoA from E. coli

PCR cloning Genosys

P20 CATGGAGAAAATAAAGTGAAACACAGC

N terminus of full length phoA from E coli.

PCR cloning Genosys

P69 GAGAGAGAACTAGTGGAGGAAGAAAAAATGAGTAAAGGAGAAGAAC

SpeI GFPuv forward primer

PCR Genosys

P70 ACCGACTAGTAGGCCTATTATT

SpeI GFPuv reverse primer

PCR Genosys

P79 CCATTACCTGTCCACACAATCTGCCC

SalI site directed mutagenesis (sdm) for GFPUV bp884-907

sdm Genosys

P80 GGGCAGATTGTGTGGACAGGTAATGG

SalI sdm for GFPUV bp884-907 reverse

Sdm and PCR Genosys

P81 CGATTAATTAAGTCGACATCTAGAGTTTAAACTTTAATTAAGCCCGGGCTGCA

Linker for pOT1 with ClaI-PstI ends and internal SalI

LINKER Genosys

P82 GCCCGGGCTTAATTAAAGTTTAAACTCTAGATGTCGACTTAATTAAT

Linker for pOT1 with ClaI-PstI ends and internal SalI, compliment of P81 minus overhanging ends

LINKER Genosys

P86 GCTTCGCAACGTTCAAATCCGC

mapping primer for Pharmacia transcriptional terminator

PCR Genosys

pOT-reverse

CATTTTTTCTTCCTCCACTAGTG

cy5 label for sequencing inserts in pOT1

SEQUENCING Pharmacia

36

2.4 Growth conditions

2.4.1 Growth media

Rhizobium were grown at 27°C on TY medium with CaCl2 (6mM) (Beringer, 1974)

or acid minimal salts (AMS) based on Brown and Dilworth (Brown & Dilworth,

1975) with the following adaptations: potassium phosphate (0.5mM), MgSO4

(2mM), CaCl2 (0.17mM) and MOPS buffer (20mM, pH 7.0). Minimal media was

supplemented with either glucose (10mM) or succinate (10mM) and ammonia

(10mM) Agar was added to media at 1.5% (Difco Bitec). E.coli were grown at 37°C

in Luria-Bertani broth (LB) consisting of tryptone (10 g.l-1), yeast extract (5 g.l-1),

NaCl (5 g.l-1). Agar was added to the media as necessary (Difco, Bitek: 1.5%).

Strains were routinely stored at -80°C in 15% glycerol after snap freezing in liquid

nitrogen.

2.4.2 Antibiotics

Media was supplemented with antibiotics where appropriate. The routine

concentrations are stated below with variations stated in the text:

37

Antibiotic Rhizobium E. Coli

Streptomycin 500 µg.ml-1 25 µg.ml-1

Kanamycin 40-160 µg.ml-1 25 µg.ml-1

Tetracycline 5 µg.ml-1 (TY)

2 µg.ml-1 (AMS)

10 µg.ml-1

Gentamycin 20 µg.ml-1 10 µg.ml-1

Ampicillin - 50 µg.ml-1

Trimethoprim - 10 µg.ml-1

Spectinomycin 100 µg.ml-1 50 µg.ml-1

2.4.3 Microtitre plates

For the library construction (Chapter 5) Rhizobium strains were grown in 96 well,

flat bottom microtitre plates (Iwaki, Japan). Growth medium was AMS (200µl) the

plates were incubated at 27°C, 125 rpm in a custom built holder mounted on a New

Brunswick gyratory shaker. The holder built in the departmental workshop by Mr A.

Dawson, consists of an open toped aluminium box with two walls that can be

adjusted to hold the plates in place. The plates are incubated in batches of 28 plates

as this number fills one layer of the box. A maximum of 5 layers (140 plates) can be

incubated in this way.

Microtitre plates were assayed using a Biolumin960 plate reader (Molecular

Dynamics) controlled by an Apple PC. Plates were assayed in batches of 28 plates,

for optical density (630nm) and fluorescence (405/10nm excitation, 505/10

emission). The data was analysed using an Excel spreadsheet. Wells with OD630

38

below 0.15 were excluded. For the remaining wells, the relative fluorescence, V,

was calculated according to the equation:

V=F-X/OD630-Y

Where F is the measured fluorescence, X is the average fluorescence for a blank

uninoculated well on each plate, OD630 is the optical density for a particular well and

Y is the average OD630 for the blank wells.

2.4.4 Stains and fluorescent observations

Phosphatase activity was detected on plates by supplementing them with 5-bromo-4-

chloro-3-inodolyl phosphate (XP)(50µg ml-1)(Sigma). Samples for microscopic

study were stained with ELF stain (1:20 dilution)(Molecular Probes inc.)

Observation of colonies expressing GFP and GFPuv were made using a

transilluminator (UVP model TL-33E) fitted with four 20nm bulbs and a long wave-

length emission filter

2.5 Molecular Biology

2.5.1 DNA Isolation

2.5.1.1 Plasmid preps

Routine plasmid extractions were carried out using alkaline lysis as previously

described (Sambrook et al., 1989). Some plasmids were prepared using the boiling

39

method described by Arnold et al. (Arnold & Pühler, 1988). For sequence analysis

and other high quality plasmid DNA isolations, FlexiPrep kits (Pharmacia Biotech)

were used according to the manufacturer instructions. To obtain plasmid DNA of

suitable quality from Rhizobium, alkaline lysis was carried out and the DNA

transformed into DH5α, from where a second plasmid isolation was made.

For the PCR screening of library strains the following DNA isolation was used:

Colonies were transferred into TE (50µl) using a toothpick. The suspension was

boiled in a water-bath for 10 minutes and the cell debris removed by centrifugation

(13,000rpm 5min). For PCR amplification, 1µl of the supernatant was used.

TE is Tris-HCL (10mM, pH as appropriate), EDTA (1mM)

2.5.1.2 Chromosomal preps

Chromosomal DNA from Rhizobium was prepared as follows:

The strain was grown in TY (50 ml) with appropriate antibiotics to late logarithmic

phase. The culture was transferred to a 50 ml SS34 Sorval tube and centrifuged

(7000 rpm for 10min). The cells were resuspended in TES (4ml; 0.2 M Tris-HCl pH

7.5, 5mM EDTA, 100 mM NaCl) and the centrifugation and resuspension repeated.

Lysozyme (0.2ml; 10mg/ml in TES) and RNAase (0.04ml; 10mg/ml heat shocked)

was added and the reaction incubated (37oC, 30 min). TES (4ml), pronase (0.4ml;

10 mg/ml in TES self digested at 37o for 1 h (Sigma protease type E)) and sarkosyl

(0.24ml, 10%) were added and the reaction incubated (37oC, 30 min). The solution

40

was phenol/chloroform (Phenol:Chloroform:isoamyl alcohol, 25:24:1) extracted on

ice (Swirling) for 30 min (Equal volume of phenol/chloroform). The sample was

centrifuged (16000 rpm for 20 min) and the top (aqueous) layer collected using a

Gilson tip (1ml) with the end cut off. The phenol extraction was then repeated twice.

The aqueous layer was collected and Na-Acetate (0.1 volume, 3M) and ethanol (2

volumes, 100%) added. The clumped DNA was removed to an Eppendorf and

centrifuged to pellet the DNA. The DNA was then washed in ethanol (70%) and

dried in a vacuum dessicator, taking care not to over dry the DNA. Resuspension of

the DNA (0.75ml TE pH8.0) took place overnight (On a windmill at 4oC). The

resuspended DNA was phenol/chloroform extracted four times followed by an

extraction overnight (On a windmill at 4oC). The DNA sample was precipitated

using Na-Acetate (0.1 volume) and ethanol (2 volumes) and resuspended overnight

in TE (0.3ml, pH8.0).

2.5.2 Restriction digests

Restriction digests were carried out according to manufacturer specifications (Gibco

BRL).

2.5.3 Ligations

Ligations were carried out using T4 DNA ligase (Promega). Reactions were

routinely carried out overnight at 4°C or for 4 hr at 15°C.

41

2.5.11 Transformation

Competent DH5α cells were prepared using calcium and transformations were

carried out as previously described (Sambrook, et al., 1989) using LB in place of

SOC.

To achieve high transformation frequencies for the library construction,

Supercompetent E. coli XL2 (Stratagene) were used according to manufacturer

instructions.

2.5.4 De-phosphorylation of DNA

DNA (5-10 µg) was digested with calf intestinal phosphatase (Boehringer

Mannheim: 1 to 10 units) in 1X reaction buffer (Boehringer Mannheim) at 37oC.

After digestion the sample was phenol extracted twice. The DNA was recovered by

ethanol precipitation and resuspend in TE.

2.5.5 Removal of 5' single stranded DNA

5' DNA overhangs were filled to produce blunt ends using Klenow (1-3 units) in the

presence of dNTPs (20µM each). The reaction was incubated at 37 °C for 30min.

Termination of the reaction was achieved by heat shock (75 °C for 10min) followed

by phenol:chloroform extraction and ethanol precipitation.

42

2.5.6 Removal of 3’ single stranded DNA

Single stranded 3’ DNA overhangs were removed by reacting with T$ DNA

polymerase (Gibco BRL) according to the manufacturers instructions. Reactions

were carried out at 11°C for 20 min in the presence of dNTPs (0.33mM). The

reaction was stopped by adding EDTA (0.5M) followed by phenol:chloroform

extraction and ethanol precipitation.

2.5.7 Agarose gels and staining

Electrophoreses of DNA was routinely carried out in 0.8% agarose gel with 1X TAE

buffer (Tris-acetate (40mM), EDTA (1mM) ). The DNA was visualised by staining

in ethidium bromide and viewing under UV excitation.

2.5.8 DNA Extractions from Agarose Gels

DNA was extracted from agarose gels by excising the relative DNA band from the

gels and extracting the DNA using Prep-A-Gene (Biorad), or Geneclean (Bio 101)

according to manufacturer instructions.

2.5.9 Sequencing

DNA sequencing was carried out by the departmental (AMS) sequencing service

using the ALF Express system (Pharmacia Biotech). Plasmid DNA prepared using

FlexiPrep kits (Pharmacia Biotech) and primers were Cy5 labelled. Sequencing was

carried out using the thermal sequencing protocol.

43

2.5.10 Conjugations

Tri-parental conjugations were carried out using the plasmid pRK2013 in E. coli

strain 803 to provide transfer genes. Throughout the procedure, cell suspensions

were manipulated using pipette tips that had their tips aseptically removed.

Cultures (10ml) of helper (pRK2013) and any donor plasmids were grown overnight.

Early the next day fresh LB (10ml) with appropriate antibiotics was inoculated with

the overnight cultures (0.2 ml). These were grown for 2-3 hours at slow speed

(approximately 100rpm) until the cells were at mid-logarithmic phase. The donor

strain (1ml) and pRK2013 (1ml) were centrifuged (6500rpm, 4 min.) and the pellet

resuspended in TY (1ml). This wash stage was repeated. To prepare the recipient

strain, a slope of Rhizobium 3841 was washed in TY (3ml). The donor (0.4ml),

Rhizobia (0.4ml) and pRK2013 (0.2ml) were mixed and centrifuged (6500rpm, 4

min). The pellet was resuspended in TY (30µl) and spread on sterile filters (Hybond

N, Amersham) placed on dried TY plates and incubated (Overnight, 27°C). The

following morning the filters were removed using sterile forceps and placed in a

universal bottle containing TY and glycerol (15%). The cells were suspended by

agitation and a sample streaked on selective plates. The remaining cell suspension

was Snap frozen in liquid nitrogen and stored (-20°C).

2.5.12 PCR

Polymerase Chain reaction (PCR) amplification was carried out using Bio-x-act

polymerase (Bioline, 2-5 units) on a Omnigene thermal cycler (Hybaid). A 100µl

44

reaction mix (1x polymerase buffer) was used containing Template DNA, primer

(200ng of each), dNTP (0.2mM of each), MgCl2 (2mM) and Polymerase.

The reactions were carried out in thin walled 0.5ml Eppendorf, with mineral oil

covering the reaction solution. The reaction conditions used for library screening are

as follows:

One cycle:

Denaturation 94°C 4min

Thirty cycles:

Denaturation 94°C 45sec

Annealing 55°C 45sec

Extension 72°C 10min

One cycle:

Extension 72°C 10min

For PCR of DNA for cloning the following reaction times were routinely used:

Thirty cycles:

Denaturation 95 oC 1.5min

Annealing 55 oC 1.5min

Extension 72 oC 1.5min

One cycle:

Extension 72 oC 1.5min

45

2.5.13 Linker production

The two oligonucleotides to be annealed were mixed in equimolar amounts in

approximately 20µl TE. They were boiled for 5min in an Eppendorf with the lid on

and the reaction mix centrifuged to the bottom of the tube (Pulse). The reaction was

returned to the boiled water and allowed to anneal as it cooled to room temperature.

2.6 Pea growth

Pisum sativum cultivar Feltham First (Sutton seeds) were used throughout this

project.

2.6.1 Surface sterilisation and Germination

Peas were surface sterilised by soaking in ethanol (70%, 30 sec), washed in sterile

distilled water, soaked in sodium hypochlorite (2%, 10 min), followed by at least 3

washes in sterile distilled water. All solutions were used at volumes to cover the

peas.

The sterilised peas were placed in a petridish with sterile distilled water soaked filter

paper and allowed to germinate in the dark for between 6 and 16 hr. Growth of the

peas was carried out either in flasks or universal bottles as required.

2.6.2 Pea Growth in Flasks

Flasks (250ml) were prepared with vermiculite (100ml), cottonwool plugs and

autoclaved. The vermiculite was saturated in sterile N-free rooting solution and a

46

germinated pea placed on the surface. Flasks were inoculated with appropriate

strains (See text for details). A routine inoculum consists of the appropriate strain

grown overnight in TY or AMS with antibiotics. The inoculum was washed twice in

N-free rooting solution and inoculated onto the pea (0.5ml). The flasks, wrapped in

aluminium foil, were incubated at room temperature until the pea shoot reached the

cottonwool plug, when it was pulled above the flask neck and the plug replaced.

Growth was continued in the presence of a grow light (Sonti Agro, 16/8 hr light/dark

cycle) at room temperature until harvest (up to 5 weeks after germination). During

growth plants were watered with sterile N-free rooting solution as appropriate.

N-free rooting solution consists of:

CaCl2.2H20 1mM

KCl 100µM

MgSO4.7H2O 800µM

Fe EDTA 10µM

H3BO3 35µM

MnCl2.4H2O 9µM

ZnCl2 0.8µM

Na2MoO4.2H2O 0.5µM

CuSO4.5H2O 0.3µM

After sterilisation, the following are added:

KH2PO4 3.7mM

Na2HPO4 4mM

47

2.6.3 Pea Growth in Universal bottles

Universal bottles were filled to capacity with vermiculite and autoclaved.

Autoclaving reduces the volume of the vermiculite allowing room for a pea to be

placed on the surface of the vermiculite. Sterile N-free rooting solution (12ml) was

added along with respective inoculum and the pea covered in sterile fine sand. The

universal bottle, wrapped in aluminium foil, was placed under the grow light (as

above) and the pea incubated for up to 10 days before harvest.

2.6.4 Nodule harvesting

Nodules were harvested from pea plants after 4 weeks growth. The nodules from

root systems were collected on ice and surface sterilised. Sterilisation consisted of

soaking the nodules in sodium hyperchlorite (2%, 10 min). Three washes in sterile

distilled water were carried out. The nodules were then crushed in 10µl sterile

distilled water using a disposable pipette tip. The resulting solution was frozen (2µl

in 98µl of TY and 15% glycerol) and patched onto TY with appropriate antibiotics.

2.6.5 Rhizosphere harvesting

To harvest Rhizobium from the rhizosphere of pea plants grown in universal bottles,

individual roots (Shoot and pea removed) and vermiculite were ground in N-free

rooting solution (20ml) using a sterile pestle and mortar. The cell suspension was

filtered through a Whattman filter paper to remove large particles. The filtrate (8ml)

was centrifuged (8,500rpm, 30 sec) to remove particulate matter and the supernatant

centrifuged (13,000, 5 min) to collect the Rhizobium. The pelleted cells were

48

resuspended (50µl N-free rooting solution, 7.5% ficol) for observation and optical

trapping.

2.7 Optical Trap

For full details of the construction of the optical trap see chapter 3.

2.7.1 Microscope

A standard Nikon Optiphot epifluorescence microscope was adapted for trapping

(see Chapter 3)

2.7.2 Laser

An infrared (IR) Nd YAG laser (1064nm) was used with a He Ne laser mounted

coaxialy behind it (SL50IT; Spectron). An electrical shutter and a beam-expanding

telescope were mounted in front of the laser units. Lasers were run according to

manufacturer instructions between 0.8 and 1.2W.

2.7.3 Set Up of trap

The laser housing and microscope were mounted on a custom built rack. This brings

the horizontal plane of the laser level with the epifluorescence port and maintains

continuity between the laser and microscope. The height of the laser with respect to

the microscope can be adjusted using thin wedges below the laser supports.

49

The microscope was mounted at 90° to the normal angle of operation so that the

epifluorescence tube runs parallel to the laser housing. Adjusting the eyepiece 90° to

allow viewing of the sample accommodated this.

The laser was steered by a series of mirrors and dichroic mirrors. After emerging

from the laser housing, the beam is steered through 90° horizontal using an

adjustable IR mirror. This is mounted on the invar rails and allows both coarse and

fine adjustment in the vertical plane of the laser.

The beam enters the microscope via a custom-built port and dichroic mounting

(Micro Instruments; Longhanborugh UK) turning the beam through a further 90°

horizontally. This unit consists of a sliding mount for 3 dichroic mirrors (D1)

originally designed to allow the passage of the ultra violet (UV) excitation beam

directly through the tube whilst reflecting the laser into the same beam path. Finally

the laser was reflected vertically down onto the objective lens by a dichroic mirror

mounted on a custom built slider. A standard epifluorescence unit was mounted

above this to facilitate fluorescence microscopy.

2.7.4 Objective lens

Standard phase contrast lenses were used. For trapping a 100x oil immersion lens

with numeric aperture (NA) 1.25 was used (Nikon)

50

2.7.5 Viewing IR laser

To view the IR radiation three methods were employed. The TV and video system is

sensitive to long wavelengths allowing the sample to be safely viewed along with the

IR laser. To view the passage of the IR laser through the optics of the system, an IR

imaging device was used (Find-R-Scope; FJW Optical Systems. inc.). This hand

held electronic viewer extends the visual range of the human eye to longer

wavelengths by producing an image on a phosphorescent screen. A fluorescent

screen (Spectron) was also used to produce an image of the laser along its path.

2.7.6 Microscope Stage

A microscope stage with micrometer controls (Micro instruments) was used to allow

the fine control of the movement of trapped cells.

2.7.7 Trapping single cells

Isolation of a single cell was achieved using 0.05mm and 0.1mm path length

microcapillary slides (Camlab Ltd. UK) sealed with Vaseline as previously described

(Mitchell, et al., 1993). Flaming forceps, scalpels and glass slides with ethanol and

wiping the objective with ethanol maintain the sterility of the system. Once trapped

manipulating the focus and microscope stage moved the cells with respect to their

surroundings.

51

Chapter 3

Construction Of An Optical Trap

3.1 Introduction

Optical trapping allows the isolation of a single identifiable bacterial cell from a

population viewed by microscopy. The cell can be subsequently studied or grown in

isolation from the population in which it was derived. In the classical studies of gene

expression, cells are screened under laboratory conditions. This restricts the scope of

the study to promoter induction that can be simply modelled. The genetic response

of bacteria in natural environments where conditions are complex and cannot be

artificially created in the lab, are difficult to study. The optical trap can be used to

look at and isolate bacteria either during or immediately after exposure to a complex

environment, such as a plant root. This removes the need to screen under laboratory

conditions.

In this chapter, the design criteria and construction of an optical trap are discussed.

3.2 Construction of the Trap-Methods and Results

3.2.1 Requirements for an Optical Trap

The basic principle of optical trapping involves the refraction of light by the object

being trapped causing a momentum change to the object. To make use of this the

52

system must be designed so that the momentum change results in the object being

restrained at a defined point.

As discussed in section 1.4, several criteria are necessary to achieve trapping. The

first consideration is the light source. The collimation and coherence obtained from

laser sources is essential as the initial momentum of the photons within the beam

must have the same vector to achieve uniform momentum changes on the trapped

particle. The light intensity across the beam diameter needs to have a Gaussian

distribution to achieve maximum trapping at the focal point. This was achieved by

using a TEMOO laser profile.

The wavelength of the laser was another important consideration. A wavelength in

the infrared spectrum, 1064nm, was chosen as this wavelength is not absorbed much

by the biological material and is generally thought to be the least damaging. In

studies using traps to manipulate chromosomal structures in animal and fungal cells,

wavelengths between 700 and 100nm were compared with 1064nm. These were

found to induce less heat production by water absorption and so may be less

damaging, allowing higher trapping powers to be used (Berns et al., 1992). We used

a Nd YAG laser (1064nm) with a He Ne laser mounted coaxially behind it (SL50IT;

Spectron). The HeNe Laser produces a visible red beam that allows the IR beam to

be aimed.

In consideration of the requirements for the microscope, the objective lens used for

focusing the laser needs a high numeric aperture. The resultant acute focusing angle

achieves maximum trapping forces (Afzal and Treacy, 1992).

53

Fig. 3.1. Development of the optical trap from a, the original design to b, the final operational system. Laser path shown in red, UV path shown in blue. Additional parts added to the system are shown in hatched lines. For details see text. For full labels see Fig. 1.2.

a)

b)

Custom made Dichroic holder inserted between UV input port and UV lamp

Dichroic - reflects IR and transmits some UV - allows input of laser.

Special dichroic - reflects UV and IR, transmits visual wavelengths.

Note laser passes through optics for focusing UV, and path length correcting lens.

IR Safety filters inserted to protect user and CCTV

UV lamp and input port turned through 180°

Custom built input port and dichroic holder inserted between UV dichroic and objective - contains no pathlength correcting lens

Standard UV excitation blocks

Custom dichroic - Reflects IR, transmits UV and visual wavelengths

54

3.2.2 Development of the Trap

The initial design for the trap involved the laser entering the microscope via a port in

the side of the epifluorescence tube. It was then reflected through 90° by a dichroic

mirror that allowed the passage of the UV excitation wavelengths. The excitation

filter was mounted between this dichroic and the UV lamp. This allowed the laser

and UV to follow the same path to the objective lens being reflected by a second

complex dichroic which transmitted visible wavelengths (transmission 525 to

825nm; reflection 475 to 535nm and above 825nm) (Fig. 3.1a). This initial set up

caused several problems.

The epifluorescent tube contains a shutter for controlling the UV illumination. This

incorporated a heat filter that absorbed IR radiation. Removal of the shutter allowed

operation of the laser but prevented control of the UV and risked overheating the

cells when exposed to UV light.

When operating with this set-up, the IR image viewed on the CCTV monitor

revealed a series of diffraction rings and reflections. We were also unable to obtain a

focal point for the laser. This may have been due to the laser not filling the back

aperture of the objective, the laser being 2.4mm (1.2mm through a 2x telescope)

diameter and the back aperture being 5.5mm. Diverging the beam to fill the back

aperture of the objective was considered. A lens holder was built to hold eyepiece

lenses on the invar railings between the laser aperture and the steerable IR mirror

(Fig. 3.2). In an attempt to diverge the laser, a series of eyepiece lenses were tried at

this position (2.5x; 3.2x; 5x; 6x; 10x). The divergence of the beam was measured

55

CCTV

He Ne Laser Ne-Yag Laser 4x Telescope Electronic Shutter

Heat sink

85% IR Mirror

85%

15%

IR steering Mirror - 3D adjustable

Invar rails

Mirror housing

Neutral density filter holder

Manual shutter

IR Mirror

Visual light source

Microscope stage

Condenser lens

Objective lens

Removable Dichroic - Reflects IR, transmits UV and Visual wavelengths

UV Source

Path length correcting lens

UV controls: Iris Shutter collector lens IR

Filter

Dichroic filter block: Replaceable for different UV excitation conditions

Eye piece lens

IR safety filter Beam

splitter

Fig. 3.2. Schematic of the optical trap showing the laser path (red) UV excitation (Blue) and visual light (yellow). Note the top part of the diagram depicting the laser housing and set up is shown in plan view and the microscope is shown is side view (see photographs a and b overleaf). Not to scale.

Custom built input tube and dichroic housing inserted between UV input tube and objective

56

Fig 3.2a Photograph of the optical trapping microscope. Note the attenuation optics are housed in the silver box behind the laser input port (Right) and the laser housed in the cream box behind the UV light box (vented box left). In this photograph the standard microscope stage is shown. For optical trapping a micrometer controlled stage is used.

57

Fig 3.2b Photograph of the attenuation optics from above. This apparatus is covered during operation. The switch in the top right of the box is an interlock preventing the laser being operated if the lid is removed. The laser enters from the left (cream box) and is attenuated by reflection from the 85% reflective mirror to the heat sink (Black wedge). The working beam is reflected 90° by the steering mirror (centre) through the neutral density filters. The beam then leaves the attenuation optics (bottom) through a manual shutter (Black knob, bottom right) and is reflected through 90° into the laser input tube (bottom left).

58

crudely over a known distance by measuring the diameter of the image produced by

the red laser and by estimating the focal point by observation using the laser screen

(data not shown). None of these lenses gave an appropriate divergence. However,

by using a Phase telescope and adjusting its focal point, it was possible to obtain a

weak video image of the IR laser reflected from an IR mirror on the microscope

stage. This image still contained many diffraction rings and reflections and was too

weak to reflect from a glass slide.

Within the epifluorescence tube and dichroic housing there are two lenses. The first

is a collector lens to maximise the UV reaching the objective. The second, below the

dichroic reflecting the laser and UV to the objective, compensates for the additional

length created between the eyepiece and objective lenses by the addition of the

epifluorescence equipment. Removal of the collector lens allowed us to obtain a

focused IR beam with both 60x and 100x high NA objective lenses without having to

diverge the laser. The image was obtainable from reflections from both a glass slide

and IR mirror. Removal of the path-length correcting lens eliminated the

problematic diffraction rings. It was surmised that these were created by the

interaction of curved waveforms reflected from the lens surface interacting with the

linear laser wave fronts.

Removal of these lenses prevented the use of epifluorescence and so a new design

for the trap was considered. A new housing was constructed (Micro instruments)

based on the epifluorescence tube and dichroic mounting but without any lenses or

heat filters. This replaced the old epifluorescence tube and contains a simple

dichroic (D2) that reflects IR (1064nm) and transmits visual wavelengths and some

59

UV (transmission 425 to 975nm). The old epifluorescence tube, dichroic housing

and lamp were then mounted above. This allows standard filter blocks to be used for

fluorescence rather than having to separate the excitation filter and use a complex

and expensive dichroic for both UV and laser reflection. The two lenses and the

shutter and heat filter can also remain in place (Fig. 3.1 and 3.2).

Three minor considerations should be noted with this set up. Due to the increased

path length, there is a reduction in the excitation energy reaching the sample. This

has not created a problem as the UV lamp gives a large excess of power, which is

limited to prevent bleaching of the sample by using neutral density filters. In the

new set up, enough excitation can be achieved by reducing the power of these filters.

The new tube set up does not contain any path length correcting lenses. This results

in the objective lenses being no longer parfocal. This is only a minor inconvenience

as most of our work is carried out at 100x with little reference to other

magnifications.

Finally, the dichroic reflecting the laser does not transmit wavelengths shorter than

425nm. This means that for some excitation wavelengths, such as those for the ELF

stain, this dichroic must be removed during fluorescent observations. Again this is

not a major problem, as once a cell is identified, the excitation wavelength should be

switched off to prevent cell damage.

60

3.2.3 Alignment of the laser

The alignment of the laser was carried out empirically over a number of months. To

achieve efficient trapping the laser must fill the back aperture of the high NA lens.

The beam must also be centred on the lens so that the trap is in the field of view of

the camera. This also has a bearing on the efficiency of the trap and the creation of

equal forces on the cell.

To align the laser with the microscope, an aluminium plug was machined with

central cross hairs engraved on it. This was inserted into the side port of the first

dichroic housing. Initial coarse adjustments to the laser path were made with the fine

adjustment screws on the steering mirror (Fig 3.2) at their midpoint, by aiming the

Ne He laser onto the target cross-hairs. Vertical alignment of the laser was carried

out by raising the laser housing from the frame using a series of metal plates. These

were inserted at the corners of the housing, which was bolted through the plates to

the frame. Horizontal alignment was achieved by moving the mirror along the invar

railing. Adjustment was also carried out by rotation of the mirror on its axis (Fig.

3.3). Fine adjustment was carried out using two screws that allowed very fine

movements of the steering mirror within its mounting. The alignment was then

checked for the Ne YAG laser using the IR viewer. This sequence of alignment with

the visible laser followed by checking the IR laser was continued throughout the

alignment procedures.

61

The alignment plug was removed and the laser path opened to the objective lens. An

epifluorescence target was inserted into one of the objective mounts and the red laser

finely adjusted to the centre cross using the screws on the steering mirror. This

allows accurate alignment of the laser as the target is designed for focusing the UV

excitation beam to the centre of the objective lens. Further adjustments were carried

Fig. 3.3. Schematic diagram showing the adjustment of the laser position to fill the objective back aperture. Insert shows fine adjustment.

Coarse vertical adjustment by raising laser housing on small wedges.

Coarse horizontal adjustment by sliding mirror along invar rails

Vertical adjustment by turning the steering mirror holder.

Coarse adjustment of laser made on target cross hairs etched on plug inserted in first dichroic input port.

Dichroic aims laser into microscope when plug removed.

Fine adjustment of laser achieved by turning screws on steering mirror. These pivot mirror on both the vertical and horizontal planes.

62

out while viewing the laser with an IR viewer in place of one of the microscope

eyepiece lenses. The final positioning of the laser focal point was monitored on the

CCTV.

Whilst viewing the laser on the CCTV, the IR safety filter was remover from its

position between the epifluorescence block and the eye piece prism. This allows the

image of the laser to be observed. During normal operation of the trap, this filter is

left in place, the position of the laser having been marked on the CCTV monitor.

3.2.4 Development of the Trapping Protocol

To allow the manipulation of the single cell once it has been trapped and isolated, a

protocol was developed using microcapillary tubes (Camlab Ltd. UK). These are

optically flat capillary tubes that can be used in place of a microscope slide and

coverslip. The microcapillary tube is filled with sample using capillary action and

placed on a microscope slide with immersion oil of use under the microscope (Fig.

3.4). The microcapillaries are available with path-lengths of 0.05mm, 0.1mm and

0.2mm. To facilitate the isolation of a single cell, the microcapillary is at least three-

quarters filled with sterile broth before the sample is loaded in the remaining quarter.

The trapped cell can then be moved to the sterile broth and the tube end containing

the cell cut off using a scalpel.

Initial attempts to run the trap in the configuration described above (Fig.3.1 and Fig

3.2) resulted in bacteria and yeast cells being trapped and moved. Cells moved to the

focal point of the laser using the microscope stage controls and focus adjustment,

were trapped and subsequent movement of the stage resulted in the cell being held in

63

the centre of the field of view while the un-trapped cells moved past. The trapped

cell was held just out of visual focus due to the differences in wavelength and hence

focal depth, between the laser and the visual wavelengths.

Initial problems with cell streaming within the microcapillary tubes were overcome

by ensuring the ends of the tube were sealed with Vaseline. It was found that it was

not necessary to sterilise the immersion oil or Vaseline used on the microcapillaries

Objective lens

Immersion oil

Vaseline

Microcapillary tube

Glass slide

Sterile media Gradient of cell culture

The trapped cell can be moved up and down by focusing the microscope.

Trapping Laser

Horizontal movement is achieved by moving the microscope stage.

Once isolated from other cells, the trapped cell can be removed by cutting out a section of the microcapillary tube.

Fig 3.4. The arrangement of the microcapillary tube for optical trapping. A trapped cell in a microcapillary tube can be isolated by movement of the microscope stage. The trapped cell is cultured by cutting out the capillary section and placing it in fresh growth media.

64

as no growth occurred on LA or TY plates or in LB or TY broth when inoculated

with these compounds. Microcapillary tubes were initially autoclaved. After this

treatment however many of them stuck together and would not fill with cells. The

0.05mm tubes could not be flame sterilised as they melted however 0.1mm tubes

could be flame sterilised. Several non-sterilised 0.05mm tubes were placed in LB

and as no growth was observed, subsequent trapping was carried out using either

0.05mm tubes direct from the manufacturers packaging, using sterile forceps, or

more commonly flame sterilised 0.1mm tubes.

It was also noted during these initial trials, that cells on the bottom of the tube were

harder to trap. This had several implications to the running of the trap. Firstly if a

sample was left under the microscope for a long period of time it became

increasingly difficult to trap cells as they settled. Once on the bottom of the tube,

they were impossible to trap. Secondly, once a cell was trapped, it became more

stable in the trap if it was moved towards the top of the microcapillary tube by

adjusting the stage using the microscope focus. This allowed cells to be moved more

quickly and also avoided collisions with other cells, which were settling to the

bottom. This effect was described previously (Felgner et al., 1995).

These initial experiments were carried out with a laser output of between 1.0 and

1.4W. At these power levels, several cells could be trapped together and cells near

the trap were observed being pulled towards it.

Growth conditions for the initial attempts to culture a trapped cell are shown in Table

3.1. The section of tube containing the trapped cell was cut out using flame-

65

sterilised scalpel and forceps, placed in growth media and incubated. Non of these

conditions resulted in growth. Sections of tube cut from the microcapillary either

side of the section containing the trapped cell (fig3. 4) also gave no growth.

STRAIN GROWTH MEDIA No CELLS

IN TRAP NUMBER OF ATTEMPTED RECOVERIES

RECOVERY MEDIA

DH5α ∗ LB(Kn or Ap) 1 9 LB DH5α ∗ LB(Kn or Ap) 1 4 LB(Kn or Ap) K12 LB 1 3 LB K12 LB 3 1 LB K12 LB 1 2 BHI 3841/pIJ1687 TY (Tc) 3 3 TY 3841/pIJ1687 AMS 1 2 TY 3841/pIJ1687 AMS 1 1 AMS Table 3.1. Summary of the conditions for the attempted growth of bacterial cells isolated using the optical trap. None of these conditions resulted in the growth of the trapped cells. * These strains had plasmid pGFP, pGOT-K or pGOT-K-LacZ as markers.

The power of the laser was considered to be the most likely cause of this failure. It

was not possible to reduce the power of the laser below 1.0 W by reducing the

current to the flash tube below a critical minimum. The beam was therefore

attenuated after passing through the telescope. At the same time the telescope was

converted from 2x to 4x to give a final beam diameter of 5.2mm to fill the objective

lens back aperture.

3.2.5 Attenuation of the Laser

To attenuate the laser, a partial IR mirror was inserted into the laser path between the

laser aperture after the telescope and the steering mirror (M1). This IR mirror

reflects 85% of the laser to a heat sink, and is mounted on the invar railing using a

66

custom made mount that gives the mirror full adjustability in all directions. After the

steering mirror a custom built holder was positioned into which neutral density filters

could be placed. This holder was fixed perpendicular to the laser path (Fig 3.2 and

Fig. 3.3). This whole apparatus was enclosed in an aluminium box with black

internal surfaces and accessed through a lid interlocked to the laser control system.

Mr. A Dawson carried out the construction of these holders and box in the

department workshops.

This apparatus allowed us to control the power of the laser while maintaining its

profile and integrity (Fig. 3.5). The laser power at the sample was estimated

according to equation (1). P is the estimated power at the sample, O is the output of

the laser in Watts extrapolated from the tube current. I/I0 is the transmission value

for the neutral density filter calculated from its optical density using equation (2).

The factor 0.15 in equation 1 is the correction for the 85% mirror.

(1) P=0.15 O (I/I0)

(2) A=-log10 (I/I0)

The laser power was measured at various points along its path using different neutral

density filter combinations (Fig. 3.5). This was carried out by Jonathan Eaton (Royal

Berkshire Hospital).

Measurements were taken at the neutral density filter holder and the microscope

stage. The output of the laser as measured differed dramatically from the values

67

stated by Spectrum (Fig. 3.5a). At low input currents (13 amps) the output was

measured as only 147mW where as the stated output was 1.75W. As the input

current was increased, the measured output increased more rapidly than the stated

output so that at a current of 18 amps, the measured output was 2.22W and the stated

output was 2.66W.

The power output obtained after the 85% IR mirror had been positioned indicate that

in this set up the mirror reflects about 90% of the energy. This is probably attributed

to the angle the mirror had to be placed at to allow it to reflect to a heat sink on the

attenuation apparatus casing.

A second set of measurements taken at the microscope stage with the 85% mirror in

place gave almost identical results indicating that there is negligible loss of energy

through the dichroics directing the beam to the objective. Measurements were taken

with the objective lens removed as only a collinear beam could be measured.

The measurements taken with the neutral density filters indicate that these attenuate

the beam within the scope of their theoretical values. Fig 3.5 demonstrates that the

attenuation occurs according to the value predicted from the OD value.

Subsequent estimations of the laser output were made using the curve produced from

measuring the output with the 85% mirror in place. If neutral density filters were

used, the attenuation attributed to them was calculated according to their theoretical

absorption.

68

(a)

0

500

1000

1500

2000

2500

3000

12 13 14 15 16 17 18 19Input current I (Amps)

Out

put p

ower

(mW

)

Value quoted inSpectrum manual

Measured current atlaser output port afterthe 4x telescope

Laser Power measuredat stearing mirror afterattenuation by 85%reflective Mirror

(b)

0

50

100

150

200

250

12 13 14 15 16 17 18Input current I (Amps)

Out

put p

ower

(mW

)

Laser Power measuredat stearing mirror afterattenuation by 85%reflective Mirror

Power Laser powermeasured atmicroscope stage withobjective lens removed.

mW Power measured atmicroscope stage withOD 0.1 neutral densityfilter.



Fig. 3.5. Graphs showing the power levels for the optical trap. (a) The difference between the published power and the measured power through the telescope, along with the attenuation achieved by the 85% IR mirror. (b) The attenuation obtained using neutral density filters.

69

3.3 Isolation and Culturing of Single Cells

Trapping and isolation of single bacteria was carried out using estimated power

outputs in the tens of milli-watts range. Both E. coli and Rhizobium were trapped

and singe cells moved to the sterile broth towards one end of the capillary tube. The

segment containing the single cell was then cut out using a flame sterilised scalpel

and placed in sterile growth media (10 ml) in a universal bottle using flame-sterilised

forceps.

In the trial experiments carried out using DH5α with the plasmids pGFP, pGOT-K

and pGOT-K-LacZ. Power levels of between 6.5 and 16 mW proved too low to give

efficient isolation of the cell, taking over 10 minutes to move a cell about 5mm

necessary to ensure isolation (0.5mm.min-1). 30 mW was shown to give better

trapping speeds with both E. coli and Rhizobium (2 to 4 mm.min-1 for Rhizobium).

In the absence of viable growth after trapping, an attempt was made to establish how

long a cell remained viable in the trap. Motility is easily monitored under the

microscope and so attempts were made to trap motile cells and establish if they

remained motile after a period of time in the trap (Table 3.2). Trapping motile cells

is extremely difficult therefore the sample size is small and the laser power was

increased. It should also be noted that loss of motility does not indicate loss of

viability, just that cell damage or stress is occurring. At higher laser powers (65mW)

the cell did not remain motile. At lower laser powers (30mW) the cells escaped from

the trap due to there own motility.

70

Laser

Power

Time in Trap

(Min:Sec)

Escaped (E) or

Released (R)

Motile? Notes

30mW 0:35 E Yes Cell swam out of trap

30mW 0:22 E Yes Cell swam out of trap

65mW 1:27 R No Power too high

45mW 1:14 R No Cell only slightly motile when

trapped

45mW 0:44 R Yes Still motile when released

45mW 1:27 R Yes Cell motile when released

1:20 R No Re-trapped for further 1:20

Of particular note is the final cell in table 3.2. This cell was still motile when

released after 1min 27 sec. The same cell re-trapped 3 sec later and held for a further

1min 20 sec, was no longer motile. Although the sample size for this experiment

was small due to the difficulty of trapping motile cells, it did indicate that at 45mW

Rhizobium could remain trapped for about 1.5 min with no loss of motility, indicting

they are still viable.

Another problem noticed with these experiments was that motile cells were moving

along the tube, leading to the possibility of contaminating isolation area for the

trapped cell. Various concentrations of Ficoll in the trapping media were tested in an

attempt to increase the viscosity of the broth and thereby reduce cell motility. It was

Table 3.2. The maintenance of motility after trapping as a measurement of viability is shown. The final cell was re-trapped for a further 1:20 (arrow).

71

found that a Ficoll concentration of greater than 5% prevented motile cells from

moving rapidly.

To determine if the media within the microtitre tube and the growth broth in which

the section containing a cell was able to mix, an experiment with dye in the

microtitre tube was devised. It was thought that maybe the immersion oil that coated

the ends of the microcapillary tube when it was cut might prevent cells escaping the

tube. If the dye diffused from the tube it would indicate that any cell in the tube may

also be able to escape. Bromophenol blue (2.5mg.ml-1) was added to the Ficol and a

series of capillary tubes filled. Segments were cut out and placed in TY broth and

the dye remaining in the tube scored at 1 hr and 1 day (Table 3.3)

This indicated that mixing of the tube contents was greater from the 0.1mm

microcapillaries and that the higher percentage Ficoll also gave greater mixing.

Using immersion oil also led to greater mixing rather than preventing the tube

contents from leaking into the broth as was expected.

Using a 7.5% ficol concentration and low power leavel (30mW) obtained through the

attenuation of the laser, we obtained, from a group of 14 trapped Rhizobium, growth

from two microtitre slides. To establish if a culture grown from an isolated cell was

due to that cell or from cells drifting or swimming into the section of tube, mixed

cultures were used. The constitutively fluorescent RU1081 was mixed 1:10 and

1:100 according OD600 with non-fluorescent RU1080 (See chapter 3 for details of

these strains). At 1:100, the chances of a contaminating bacteria also being

fluorescent are low giving us reasonable confidence that if the resulting culture has

72

both the antibiotic marker as well as the fluorescence marker, that it was due to the

single isolated cell. Using this method, we demonstrated that 1 in 5 isolates were

cultured successfully under these conditions.

Ficoll

Concentrat

ion

Immersion

Oil

(Yes/No)

0.05 mm

Tube

Capillary 0.10 mm

Tube

Capillary

1 Hr 1 Day 1 Hr 1 Day

10 % Y - - - -

10 % N ++ - - -

5 % Y +/- - - -

5 % N +++ +++ -/+ -

0 % Y ++ - +++ -

0 % N +++ - +/- -

3.4 Discussion

In this chapter the construction and development of an optical trap has been

described. In the early attempts to develop the trap, the laser was inserted into the

microscope via the epifluorescence port. This was found to be unsatisfactory due to

the lenses found in this port. A custom built port was therefore designed and built.

This overcame the problems of using the epifluorescence port. It did however cause

Table 3.3. The effect on the ability of dye (Bromophenol blue 2.5mgml-1) to be released from a capillary tube containing variable ficoll concentrations. Scoring is for the amount of dye still visible in the microcapillary tube when placed in a universal containing growth media. +++ Dye still in microcapillary to - no dye in microcapillary.

73

a loss of quality of the microscope image as it disrupted the optics of the microscope.

This means the microscope no longer remains in focus when objective lenses are

changed. This has been a minor inconvenience as the microscope is dedicated to

optical trapping rather than high quality observation, and most work is carried out

using a single 100x objective.

To achieve trapping the laser had to be modified. The beam diameter was increased

to fill the back aperture of the objective lens using a 4x telescope in place of the 2x

telescope. The laser power also needed to be attenuated as its minimum setting was

too high for this work. A system of partial mirrors (85% reflection) and neutral

density filters has been used to achieve this

A protocol using microcapillary tubes has been devised to allow a single cell to be

isolated and aseptically transferred to growth broth. This has been tested to ensure

that the bulk population of cells and the isolated cell can be separated. The sterility

of the end of the tube into which the isolated cell is moved can be maintained during

its manipulation, allowing a single cell to be cultured.

Finally it has been shown that Rhizobium and E. coli can be trapped, isolated from

the bulk population and cultured. The recovery rate of trapped cells is about 1 in 5.

This value was established from a small sample size, but was confirmed in later

studies on the promoter libraries where a rate of 1 in 3 was obtained (Chapter 7).

The time taken to identify, trap and isolate a cell is a significant limiting factor in this

technique. Taking into account the set up time and sample preparation, it is possible

to trap around 12 cells in a day. However, the precision of obtaining single target

cells is unparalleled by any other technique. The ability to obtain spatial information

74

about the trapped cell is also of great significance. It would, for example, be possible

to isolate a Rhizobium from a piece of root thereby giving precise details of the

location of gene expression. The time taken to trap and isolate cells expressing

environmentally induced genes is not the rate limiting step, rather it is the analysis of

such fusions.

75

Chapter 4

Construction of promoter probe vectors

4.1 Introduction

A vector chosen for the construction of a Rhizobium promoter library must fulfil

several criteria. These include a broad replication range and suitable resistance

markers. As one objective of this work included inoculating the library into complex

environments with no antibiotic selection, it was considered that plasmid stability

was an important component. As well as the specific requirements of this project it

is also important to look at cloning procedures. The library construction was carried

out in E. coli as this organism grows fast and is easily manipulated for genetic

modification. The working library would be used in Rhizobium but considerations

for its use in other bacteria, for example Pseudomonas were made. The construction

of large representative libraries is facilitated if the plasmid is relatively easy to isolate

and manipulate. This is helped if the plasmid has a detailed restriction map or

preferably is completely sequenced.

A model of the necessary features of the promoter probe vector was devised. It had

to be a well characterised, broad host range plasmid that is stable in a complex

environment without antibiotic pressure. Antibiotic markers suitable for use in E.

coli as wall as Rhizobium needed to be incorporated in such a way as not to interfere

with the reporter genes. The nature of this work was to identify single bacteria under

the microscope. Promoterless reporter genes were needed that would enable

identification of individual bacteria under the microscope as well as colonies on agar

plates. There needs to be convenient cloning sites for genomic fragments upstream

76

of the reporter gene. The reporter section of the promoter probe vector must also be

transcriptional isolated from the rest of the plasmid. This prevents active promoters

on the plasmid inducing the reporter gene as well as the cloned promoters interfering

with the plasmids normal function.

Initially the vector pTR101 was selected. This is a RK2 (RP4) derivative and is very

stable due to the presence of the partition genes, parDE (Roberts, et al., 1990).

However, these genes made the plasmid difficult to isolate by standard laboratory

procedures, and this hindered further characterisation. We therefore tested and

subsequently used the plasmid pBBR1MCS (Kovach, et al., 1995). This plasmid has

a broad host range, moderate copy number and was easy to isolate and manipulate.

To facilitate its use for library construction, a series of new derivatives were

developed with different antibiotic resistance markers suitable for Rhizobium.

Several reporter systems were considered for this work, including gus and lacZ. Two

reporter systems were studied in more detail to determine there suitability for the

detection of gene expression in single cells. The first, phoA is a periplasmicaly

expressed enzyme, whose expression can be detected in whole singe cells under the

microscope using the fluorescent stain ELF (Molecular probes inc.). It can also be

detected on plates using 5bromo-4-chloro-3-inodolyl phosphate (XP). The second

uses a protein derived from the jellyfish Aequorea victoria (Chalfie et al., 1994;

Inouye & Tsuji, 1994). This protein, known as Green Fluorescent Protein (GFP), has

intrinsic fluorescence when exposed to UV light, which can be detected in single

cells.

77

4.2 Methods and Results

4.2.1 Characterisation of reporter genes for use in IVET-OT

A number of reporter genes were considered for the vector system. A reporter gene

for use in IVET-OT should enable the isolation of non-constitutive promoters and the

identification of those promoters when active. The reporter also needs to be

detectable on plates as well as in single cells under the microscope.

To produce an IVET vector in which selection against expression occurred, we

attempted to clone the SacRB gene. In this strategy we were attempting to select

against constitutive promoters. This gene produces an enzyme, which breaks down

sucrose producing a lethal product and can therefore be used as a negative expression

marker (Gay et al., 1985; Gay et al., 1983; Ried & Collmer, 1987). The sacRB gene

was cloned into pGEM-T as a 1.45Kb PCR fragment from pWS233 to give pRU162.

Attempts were made to clone sacRB into the SmaI site of pBluescript SK as a

NotI/NcoI infilled fragment. Thirty-five white colonies were obtained from two

ligations but restriction mapping indicated that in all cases the insert was cloned in

the wrong orientation; reading towards the lacZ promoter so it would not be

expressed. This indicates that expression of sacRB at high levels may be deleterious

even in the absence of sucrose in E. coli. This was confirmed by attempting the

cloning into pBluescript KS. Again clones were only obtained reading towards the

promoter. Intolerance to sacRB under the lacZ promoter could be due to the high

copy number of the bluescript plasmid. Although generally the sacRB genes would

not be expressed as highly in the final constructs, we were concerned that a stray

78

environmental promoter might cause toxicity due to its expression level. For this

reason, further work on sacRB was discontinued.

4.2.2 Construction of pPOT and pGOT vectors

The pPOT and pGOT vectors were designed as standard promoter probe vectors. A

number of important features were incorporated. A modular design was used

allowing different reporters and antibiotic resistance genes to be used. Initially

promoterless phoA was cloned as the reporter.

4.2.2.1 Cloning of Omega Transcriptional Terminators

For the promoter probe to operate correctly the reporter must be silent except when

influenced by the cloned promoter. To prevent promoters in the rest of the plasmid

effecting the reporter a transcriptional terminator (TT) was used. A second

transcriptional terminator was positioned after the reporter to prevent transcription

continuing into the rest of the plasmid and effecting its function.

The transcriptional terminator flanking the omega spectinomycin cartridge on the

plasmid pHP45 was amplified by PCR using the primers P12 and P13. This was

cloned into pGEM-T in both orientations giving pRU160 and pRU161. The

orientation of the cloned fragment was confirmed by restriction mapping and the

BamHI site in pRU161 removed by digestion with BamHI, followed by infilling the

cleaved ends and religating them (Fig. 4.1). This was done as BamHI was to be used

as the cloning sits for inserting promoters and so other BamHI sites needed

removing.

79

4.2.2.2 Cloning of phoA reporter gene

The reporter phoA was considered appropriate for this project as it was easily

monitored on both plates and in single cells. It was also sensitive enough to give an

pHP45

PCR (P12, P13)

Cloned into pGEM-T

pHP45

PCR (P12, P13)

Cloned into pGEM-T

SacII

BamHI SpeI PstI

NotI

PRU161PRU160

ApaI

SacII BamHI

SpeI PstINotI ApaI

Fig. 4.1. Schematic of the construction of pRU160 and pRU161. See section 4.2.2.1 for details. (Not to scale)

Transcriptional terminator

80

indication of the activity of promoters. In keeping with the strategy to make the

promoter probe vector modular, this reporter was cloned as a cassette. GFP was also

inserted into the vector in place of phoA to give a second reporter system.

Using primers P19 and P20, phoA was amplified by PCR from E. coli strain K12.

The forward primer was located close to the 5' end of the gene, including the ATG

but not the promoter. To produce a cassette, the PCR fragment was cloned into the

blunt SmaI site in pJQ254. This site lies between two NotI sites thereby allowing the

reporter to be inserted as an easily replaced cassette. This procedure can be used for

any sequenced reporter gene. To provide the upstream (read out) transcriptional

terminator it was cloned as a NotI cartridge upstream of the transcriptional terminator

in pRU161 with the infilled BamHI site (Fig. 4.2). The orientation of phoA was

confirmed by restriction mapping.

The transcriptional terminator and phoA were cloned into pSK as a SacII/NotI

(partial) fragment creating pND-1.

To facilitate insertion of the upstream transcriptional terminator a marker gene was

used. The plasmid pRU279 was constructed by cloning the kanamycin resistance

(KnR) gene from pRAT1 as a BamHI fragment downstream of the TT in pRU160

(Fig. 4.2). The kanamycin resistance gene could then be used to identify the

transcriptional terminator. The TT-KnR fragment was cloned upstream of phoA in

pND-1 as a PstI ApaI fragment to create pND-2.

81

pRAT1

PhoA PCR fragment from E.coli K12 using primers P12 and P20

pJQ254

SmaI

NotINotI

SacII BamHI SpeI PstINotI

BamHI site removed

PhoA cloned into pRU161 (with infilled BamHI site) as NotI fragment

PhoA

NotI

KpnI

pND-1

SacII/NotI (Partial) Fragment cloned into pSK

BamHI fragment

KnR Cloned upstream of TT in pRU160

KnR

ApaI

pRU160

SacIIBamHI

SpeI PstI

NotI

pRU279

KnR

ApaI PstI

SacII SpeI NotI

PhoA

NotI

SacI BamHI

PstIXbaI

ApaI

ApaI/PstI fragment cloned into pND-1

SacII SpeI NotI

PhoA

NotI

SacI BamHI

PstIXbaIApaI

KpnI

KnR

pND-2

SacII

Fig. 4.2 Schematic of stages in the construction of pPOT and pGOT plasmids. See section 4.2.2.2 for details. (Not to scale)

82

4.2.2.3 Cloning of antibiotic markers.

The chloramphenicol (CmR) resistance of the original pBBR1MCS is not suitable for

use in Rhizobium. This gene was therefore replaced with either spectinomycin (SpR)

resistance or kanamycin resistance genes to enable its use in Rhizobium under

various conditions. These genes were inserted in a cassette to enable them to be

changed, extending the use of the vector.

The spectinomycin cartridge from pHP45 Omega was cloned as an infilled HindIII

cartridge into the DraI sites in the CmR gene of pBBR1MCS (Fig. 4.3), deleting the

CmR gene. The SpR cassette included transcriptional terminators thus preventing it

effecting the plasmid transcriptionaly. The SacII fragment from pND-2, containing

KnR and phoA between the same two transcriptional terminators was cloned into the

SspI sites in the MCS and lacZ gene of pBBR1MCS by removing the 3' overhanging

ends of the SacII sites using T4 DNA polymerase. This deleted most of the MCS

and lacZ gene. Finally the KnR marker, used as a cloning marker, was removed as a

complete BamHI deletion to give plasmid pOTS.

A kanamycin resistant version, pOTK, was simultaneously constructed by cloning

The KnR gene from pHP45 Ωkan as an infilled HindIII fragment into the DraI sites

in CmR gene of pBBR1MCS and proceeding as for the spectinomycin resistant

version above. The upstream transcriptional terminator was inserted without the KnR

marker and successful insertion identified by restriction mapping.

83

pND-2

Spectinomycin resistance cartridge from pHP45 SpR

HindIII HindIII

1) HindIII sites infilled to produce blunt ends.

pBBR1MCS

CmR

DraI

MCS LacZα

SspI

SspI

SacII SpeI NotI

PhoA

NotI

SacI BamHI

PstXbaI ApaI

KnR

KpnISacII

3) SacII sites digested to give blunt ends

2) Cloned into DraI sites

4) Cartridge cloned into SspI sites removing most of the MCS

BamHI BamHI

5) KnR removed by BamHI digest.

Fig. 4.3. Final stages in the construction of pPOT. See section 4.2.2.3 for details. (Not to scale)

84

4.2.2.4 Introduction of Multiple Cloning Sites

The plasmids constructed had few restriction sites due to the deletion of most of the

multiple cloning site (MCS). BamHI was the only site available for cloning in front

of the reporter gene and this site was to be used to clone genomic fragments. To

make the plasmid adaptable and to allow further cloning a new MCS needed to be

introduced into the BamHI site and result in the recreation of only one BamHI site.

The restriction enzyme BglII has compatible ends with BamHI however the

recognition site is different thus it can be ligated to a BamHI site without recreating

the BamHI site. The MCS of pMP220 has both a BamHI and a BglII site but has a

limited number of useful restriction sites. Cloning the BamHI/KpnI fragment from

pSK MCS into the BamHI/KpnI sites of pMP220 MCS and subsequently cloning the

BamHI/BglII fragment created into the BamHI of the promoter probe vectors gave a

useful MCS (Fig 4.4).

85

4.2.2.5 Insertion of GFP reporter gene.

A new fluorescent reporter, Green fluorescent Protein (GFP) became available on the

market (Clontech) (Chalfie, et al., 1994). The gfp gene codes for a protein that is

fluorescent when excited by UV light (Absorption maximum peak 395nM, minor

peak 470nm. Emissions maximum peak 509nm, shoulder at 540nm (See Fig.4.5).

The protein was originally isolated from the bioluminescent jellyfish Aequorea

victoria, where light is produced by the transfer of energy from Ca2+-activated

XbaI

SpeI NotI

PhoA

NotI BamHI

SpeI

Bgl

II *

Ec

oRI

Cla

I Sa

cI

Kpn

I Ap

aI

XhoI

Sa

lI C

laI

Hin

dIII

Ec

oRV

Ec

oRI

PstI

SmaI

Ba

mH

I

KpnI/BamHI fragment from pSK (62bp) cloned into KpnI/BamHI of pMP220.

BglII/BamHI fragment cloned into BamHI to give final MCS

Fig.4.4. Insertion of the multiple coning site to produce the plasmid pPOT. See section 4.2.2.4. (Not to scale). * This BglII site does not occur in the final MCS as it is ligated into a BamHI site.

86

photoprotein aequorin to GFP. The GFP chromophore consists of a cyclic tripeptide

derived from Ser-Tyr-Gly in the protein sequence (Cody et al., 1993).

This is only fluorescent when located in the complete protein. When cloned into

bacterial cells this fluorescence is visible in colonies on an UV transilluminator and

is visible under the microscope in single cells. No cofactors, substrates or other gene

products are needed for fluorescent production.

GFP has the advantage over phoA that it does not require the addition of a substrate

and an enzymatic reaction to occur before it can be visualised. Using phoA also had

the disadvantage that its action was often obscured by native phosphatases in the

plant roots and Rhizobium making microscopic studies particularly difficult. GFP

fluoresces at a wavelength where there is less autofluorescence of other biological

material.

Plasmid pGOT-S and pGOT-K were created by replacing phoA with wildtype gfp as

a BamHI/SpeI cartridge from pGFP (Clontech). Plasmid pGOT-K was conjugated

into 3841 and designated strain RU1080.

Fig. 4.5. The absorption (Dotted line) and emission (solid line) spectra of wt.GFP. The two high peaks are for EGFP, a red shifted GFP optimised for use in Eukaryotes. From Clontech product protocol Living colours™, GFP application notes (PT2040-1)

87

4.2.3 Cloning of test promoters in pPOT and pGOT vectors

To test the characteristics of the promoter probe vector and the reporter genes, a set

of promoters were cloned into the MCS. The lacZ promoter (placZ) from

pBluescript is constitutivley expressed at high levels in R. leguminosarum. The dctA

promoter (pdctA) and nodC prompter (pnodC) are induced by dicarboxylic acids and

hesperatin respectively, enabling the sensitivity of GFP to be determined under a

range of transcriptional activation.

4.2.3.1 Cloning the Lac Z Promoter

The lacZ promoter from pSK was cloned as a 1Kb Sau3A fragment into the BamHI

site of pGOT-K and pGOT-S. Correct orientation was determined by induction with

IPTG. Fluorescent colonies were identified using an UV transilluminator optimised

for GFP fluorescence. Restriction mapping confirmed the correct orientation of the

lacZ promoter. These constructs were named pGOT-K-lacZ and pGOT-S-lacZ

respectively. The plasmid pGOT-K-lacZ was conjugated into 3841 and designated

RU1081.

4.2.3.2 Cloning of the dctA Promoter

The dctA promoter was cloned from pRU61 as a 1KB BamHI/XhoI fragment into

pGOT-S and pGOT-K. This fragment includes the divergent promoters for both

dctA and dctB. In plasmids pGOT-K-dct and pGOT-S-dct the promoter from dctA

acts towards gfp. This orientation was confirmed by restriction mapping. The dctB

promoter is transcribed at a constitutive low level but is induced up to 20 fold by

88

dicarboxylates (Reid and Poole, 1998). Strain RU1087 was created by conjugation

of pGOT-K-dct into 3841.

4.2.3.3 Cloning of the nodC Promoter

The nodC promoter was cloned from pIJ1814 as a 0.35Kb BamHI fragment and

inserted into the BamHI cloning site. The correct orientation was determined by the

ability to induce fluorescence with hesperitin. The plasmid was conjugated into 3841

and designated strain RU1088.

4.2.4 General fluorescence of pGOT vectors

The fluorescent levels for the three vectors with cloned promoters were compared on

plates and in broth when expressed in Rhizobium. Consistent results for fluorescence

were difficult to achieve and in comparison to fluorescence achieved later with

GFPuv, the fluorescence level was very low. High levels of fluorescence on plates

and under the microscope were only achieved with the placZ. Induction of the

pnodC by growth on 1µM Hesperitin gave very low fluorescence levels and

fluorescence was never achieved during induction of pdctA by growth on succinate

For the fluorescent protein to be produced, GFP requires oxygen. Only after

exposure to sufficient oxygen can fluorescence be seen as molecular oxygen is

necessary for the formation of the mature chromophore (Heim et al., 1994). We

tested whether this was causing the low fluorescent levels by growing cultures of

pGOT-K-LacZ in E. coli and Rhizobium in universal bottles (10ml broth) and in 1l

flasks (50ml broth). E. coli cultures incubated in universal bottles occasionally gave

89

low fluorescence levels and often did not fluoresce. These cultures had to be grown

in large flasks with maximum aeration to give fluorescence. On plates, the colonies

had to be grown for long periods and left for up to several days at 4°C to achieve

visible fluorescence. When the plasmid was in Rhizobium better results were

obtained, probably due to the longer growth time allowing the protein to mature into

the fluorescent form. Colonies on plates were generally fluorescent but still needed

an overnight incubation at 4°C to achieve maximum fluorescence.

4.2.5 Stability of pGOT vector

An important aspect of the vector design is the ability to expose the final library to

complex environmental conditions and still maintain the plasmid with no antibiotic

pressure. The strains RU1080 and RU1081 were therefore compared for

fluorescence expression after inoculation on plants. These strains were compared as

RU1081 contains the pGOT-K plasmid with the LacZ promoter and constitutavely

expresses GFP, whilst RU1080 has plasmid pGOT-K with no promoter.

The stability of the plasmid in Rhizobium, when infecting roots was determined. Pea

plants were grown in flasks and inoculated with RU1080 and RU1081 using our

standard plant growth methods (see Section 2.6). Control plants were either left

uninoculated or inoculated with Rhizobium strain 3841 to act as positive and negative

controls for nodulation. Once grown, the nodules from root systems were collected

and surface sterilised. They were then crushed in 10µl sterile distilled water using a

disposable pipette tip. The resulting solution was frozen (2µl in 98µl of TY and 15%

90

glycerol) and patched onto TY (Streptomycin 500µg ml-1) and TY (kanamycin 40µg

ml-1).

A total of 128 nodules from RU1080 gave growth on TY (Streptomycin) of which

only 1 grew on TY (kanamycin). Of the 122 nodules, from RU1081 that grew on TY

(Streptomycin), only 5 grew on TY (kanamycin). The 5 colonies that grew from

RU1081 nodules were tested for fluorescence. Only 3 of the colonies that grew on

TY (kanamycin) were fluorescent.

From this it was apparent that there were stability problems during nodulation of

plants. It appeared that sections of the plasmid, including parts of the KnR gene and

either placZ or gfp were being deleted. To determine the degree of instability a

further test was carried out in vivo. This involved maintaining growth in liquid

culture for 5 days (approximately 30 generations) with no antibiotics, and monitoring

the maintenance of the plasmid.

Two 1l flasks (50 ml AMS, glucose 10mM, ammonia 10mM) were inoculated with

each culture (RU1080 and RU1081). Kanamycin (40µg.ml-1) was added to one flask

for each strain to maintain the plasmid. The inoculum was a 4-day growth of the

strain on a TY slope (Kn. 40µg.ml-1). The slope was washed twice in 5 ml AMS and

500µl used to inoculate the flask. A sample of the inoculum (1ml) was snap frozen

in glycerol.

The cultures were grown overnight (26°C, 250rpm) and a sample (1ml) taken and

frozen in 15% glycerol. The cultures were then sub-cultured into 50ml of fresh

91

broth, and growth continued. The inoculum was calculated to give an OD600 of 0.2

by 6pm when the sampling and sub-culturing was repeated. This was continued for 5

days, sampling at 10am and 6pm.

Once all the samples had been collected, they were spread on TY plates

(Streptomycin 500µg.ml-1) and once grown, patched onto TY (Streptomycin

500µg.ml-1) and TY (Kanamycin 40µg.ml-1). Initial observations of the colonies as

they grew indicated that there were several time periods that occurred. Large

colonies grew in about 3 days, followed about 1.5 days later by slower growing

colonies. A background of pin colonies was also observed on many plates. The fast

and slow growth occurred in both the RU1080 and RU1081, initially grown with and

without kanamycin, indicating that expression of GFP was not necessary but that at

least part of the plasmid was being maintained. For strain RU1081, fluorescence was

observed in the slow growing colonies but was generally absent in the fast growing

colonies. All the colonies from RU1080 were non-fluorescent as expected. The

patch plates indicated that for both RU1080 and RU1081, kanamycin resistance was

maintained whether or not kanamycin was present in the broth cultures and is not

dependent on the speed of growth of the colonies recovered. The original inoculum

also showed these faster and slower growing colonies with similar fluorescent

pattern. It appeared therefore that the plasmid was being maintained whether or not

kanamycin was present, but at the expense of a deletion of part or of the entire

reporter region. Loss of this region was allowing clones to grow at a faster rate.

When the frequency of the occurrence of the fast growing colonies was plotted a

steady increase occurred throughout the experiment (Fig 4.6). The frequency

92

increased at the same rate in the culture with no kanamycin, however after three days

it appears to drop to zero. This may be due to the deletion of the kanamycin gene but

the sample size was very small so this conclusion can not be confirmed. The

inoculum also had a number of fast growing colonies indicating the deletion was

occurring rapidly and under normal growth conditions.

Clearly these complex results are not easy to account for without detailed molecular

analysis of many plasmids. However, this seemed of little real value to the central

aims of this project. Instead a general conclusion was made that while the basic

replicon is stable in vitro, deletions appear to be occurring in vitro that effect the

expression of gfp. These putative deletions were not found in E. coli DH5α that is

recA-. However, the presence of two colony types, indicative of deletion, did not

require a promoter, indicating that the deletions may be due to recombination events

probably between the four copies of the omega transcriptional terminator.

Fig. 4.6. Comparison of the occurrence of fast growing colonies (Non-fluorescent and postulated to have deleted promoter-reporter region) in RU1081 over 4 days when grown in the presence and absence of kanamycin. All the clones maintained there kanamycin resistance.

02468

101214161820

Inoc 1-am 1-pm 2-am 2-pm 3-am 3-pm 4-am 4-pm

Sample (Day - Time)

perc

enta

ge fa

st g

row

ing

colo

nies NO kn

Plus kn

93

4.2.6 Construction of pOT1 vector

Due to the stability problems and the low fluorescence levels in the pGOT vector, a

new plasmid, pOT1 was developed. The promoter probe pOT1 was based on the

vector pBBR1-MCS-5 (Kovach, et al., 1995), the reporter gene gfpuv and uses two

different transcriptional terminators.

The reporter gene gfpuv was developed using in vitro DNA shuffling to introduce

point mutations (Crameri et al., 1995). Three amino acid substitutions were made

(Phe-99 to Ser, Met-153 to Thr, and Val-163 to Ala. Numbering according to

wtGFP) which resulted in an 18 fold increase in fluorescence while retaining the

excitation and emission maxima of wtGFP (Fig 4.7). Significantly, in E. coil, GFPuv

is expressed as soluble, fluorescent, protein under conditions were the majority of

wtGFP is expressed in non-fluorescent inclusion bodies. GFPuv is also partially

optimised for use in prokaryotes by replacing rarely used codons for those preferred

by E. coli.

The plasmid pBBR1-MCS-5 was newly available. It uses gentamycin resistance as a

marker, which is suitable for use in Rhizobium. This removed the need to clone a

new antibiotic resistance marker, removing the need for additional transcriptional

terminators. The reporter gene and cloning sites were transcriptionaly isolated from

the rest of the vector by unique transcriptional terminators, to reduce the risk of them

interacting. This meant the new vector would have two heterogeneous

transcriptional terminators rather than four homologous ones. A bacterial ribosome-

binding site was also incorporated to optimise fluorescence as it was noted that the

94

existing site in pGFPuv was not suitable (see Fig 4.9). As in the pGOT and pPOT

vectors, the plasmid was constructed in modular form to enable adaptations, as new

or more convenient reporters become available.

The gfpuv gene was amplified by PCR from the vector pGFPuv using the primers

P69 and P70 and cloned into pGEM-T. The reverse primer, P70 included the SpeI

cloning site in gfpuv. P69 extends over the start site for GFPuv and incorporates a

ribosome binding site, GGAGGA, and a SpeI restriction site (see Fig. 4.8). In

pGEM-T the SalI site in gfpuv was removed by site directed mutagenesis (Stratagine

Quick change Kit, as per manufacturers instruction) using primers P79 and P80.

This site had been added during the construction of pGFPuv. We mutated the bases

back to the wildtype sequence to allow a new promoter cloning strategy to be used.

This involves cloning genomic fragments into a partially filled SalI site in the MCS

(see chapter 5). The change was confirmed by sequencing on both strands of DNA

using primers P97 and P100.

Fig. 4.7 Comparison of the excitation (dotted lines) and emission (solid lines) spectra for wtGFP (Black, low peaks) and GFPuv (Grey, high peaks). From Clontech product protocol Living colours™, GFP application notes (PT2040-1)

95

To reduce the possibility of recombination a transcriptional terminator with a

different sequence to the omega transcriptional terminator was selected. The

transcriptional terminator from pKK232-8 (Pharmacia) was inserted upstream of

gfpuv as an EcoRI clone. The orientation of the TT was determined by PCR

screening using primers P69 and P86. The gfpuv gene and TT were cloned into

pOTK as a SpeI cassette, replacing the phoA cassette to create pND-4 (Fig 4.9).

To insert the new reporter gene with the omega and Pharmacia transcriptional

terminators into pBBR-1-MCS-5, a complex double partial digest was necessary. A

partial NcoI digest and isolation of the 5.7Kb fragment of pND-4 results in an NcoI

site at the 5' end of the reporter cassette. A partial StuI digest and isolation of the

1.182Kb fragment results in a StuI site (Blunt) at the 3' end of the reporter fragment

(Fig 4.10). This was cloned into the NcoI and SspI (Blunt) sites of pBBR-MCS-5,

deleting 0.7kb to produce the plasmid pBB-Gm-GFPuv.

met GAGAGAGAACTAGTGGAGGAAGAAAAAATGAGTAAAGGAGAAGAAC Fig.4.8. The sequence of primer P69 showing the SpeI site (Italic underlined), Ribosome binding site (Bold) and ATG start codon (over lined) with translation.

96

P70 P69

PCR product from pGFPuv Using P69 and P70 - Creates RBS and speI site at 3' end

Cloned into pGEM-T

SalI site removed

TT from pKK232-8 inserted as EcoRI fragment

Fig. 4.9. Stages in the construction of pOT plasmid. See section 4.2.7 for details. (Not to scale)

XbaI SpeI

NotI NotI BamHI

SpeI

phoA

SpeI phoA cartridge in pOTK replaced by speI gfpuv cartridge.

pOTK NcoI

gfpuv

SpeI SpeI

RBS

EcoRI

SalI NcoI

gfpuv

SpeI SpeI

RBS EcoRI

Stop CodonSalI

Nco1

SpeI

BamHI

SpeI

NcoI

gfpuv

RBS

EcoRI

SalI NcoI

Omega TT

97

SpeI

BamHI

SpeI

gfpuv

RBS

EcoRI

NcoI

NcoI NcoI

NcoI

gfpuv

SpeI

StuINcoI

Final fragment cloned into pBBR-1-MCS-5

StuI

knR

First partial fragment recovered (NcoI) 5.7Kb

Second partial digest (StuI) and isolation of 1.182Kb fragment. This fragment is cloned into pBBR-1-MCS-5

Fig. 4.10. Schematic showing the two partial digest fragments from pND-4 used to clone the reporter cassette into pBBR-1-MCS-5. (Not to scale)

Enlargement of section of plasmid

98

In the pGOT plasmids an MCS was introduced by cloning an existing MCS from

other plasmids. Although this resulted in a workable MCS, it was not ideal. To

facilitate the removal of any DNA inserted into the new plasmid, a set of rare cutting

restriction sites surrounding the SalI site should be included. This allows genomic

DNA to be inserted into the SalI site using the partial in-fill method (See Chapter 5)

whilst allowing it to be removed using the rare cutters which are unlikely to be found

in the inserted fragment. To allow this ideal MCS to be constructed, two

complimentary linkers, P81 and P82 were produced (Constructed by Genesis) and

annealed by heating (Fig 4.11). The ends of this linker had overhanging ends

complimentary to DNA digested with PstI and ClaI. This allowed the linker to be

cloned into the plasmid pBB-Gm-GFPuv to create the final pOT1 plasmid.

Plasmid pOT1 has a modular construction allowing it to be adapted. Table 4.1

indicates the important restriction sites and their function. The location of the sites

can be seen in the maps of the MCS (Fig 4.12) and the whole plasmid (Fig 4.13).

The cloning of genomic DNA into the SalI site is described in chapter5. Its re-

isolation for subsequent characterisation is facilitated by the inclusion of the rare

PacI SalI XbaI PmeI PacI SmaI ------ -------- ------ -------- ------ -------- P81 CGATTAATTAAGTCGACATCTAGAGTTTAAACTTTAATTAAGCCCGGGCTGCA P82 TAATTAATTCAGCTGTAGATCTCAAATTTGAAATTAATTCGGGCCCG ~~~~ ~~~~~ ClaI complimentary end PstI complimentary end Fig. 4.11. The complimentary linkers P81 and P82 were annealed and cloned to produce the MCS for pOT1, introducing the restriction sites shown.

99

cutter, PacI and PmeI. The two XbaI sites can also be used. Another feature of the

MCS is the inclusion of a unique PstI site with SmaI sites on either side. This allows

the inclusion and removal of a second reporter gene.

Restriction

Enzyme

Number

of sites

Proposed use of sites

EcoRI 2 Pharmacia transcriptional terminator cassette

EcoRV 2 Cassette including gentamycin resistance gene and Omega transcriptional

terminator

BamHI 2 Removes most of gfpuv cassette

SacI 1 3' end of gfpuv cassette

SpeI 1 5' end of gfpuv cassette

PstI 1 Site for cloning of second reporter

SmaI 2 Sites either side of PstI. Allow removal of DNA cloned in PstI

SalI 1 Site for cloning of genomic fragments

PacI 2 Sites for removal of fragments cloned in SalI

XbaI 2 Alternative sites for removal of fragments cloned in SalI

PmeI 1 Unique rare cutter - can be used in conjunction with PacI for fragment

retrieval from SalI clones

ClaI 1 Unique cloning site in MCS

All the parts of pOT1, which were not present in pBBR1-MCS-5, were sequenced.

This allowed the entire sequence of pOT1 to be deduced (Fig. 4.13).

Table 4.1 Summary of the important cloning sites in pOT1 and their function. The location of sites can be seen in figs. 4.12 and 4.13.

100

_SalI__ EcoRV_ __PacI__ PmeI __PacI__ 5'CGGATCTAGATATCGATTAATTAAGTCGACATCTAGAGTTTAAACTTTAATTAAG XbaI ClaI XbaI PstI__ SmaI__ SmaI__ SpeI__ CCCGGGCTGCAGCCCGGGGGATCCACTAGTGGAGGAAGAAAAAATGAGTAAAGGA 3' BamHI Fig.4.12 The multiple cloning site in pOT1. Unique sites are shown in bold.

gfpuv

Ω TT

Cloning site for genomic DNA

Rare cutting sites for extraction of genomic fragment

Sites for extraction of genomic fragment

Sites for insertion and removal of second reporter

101

Fig. 4.13 pOT1 plasmid.

pOT15278 bp

gfpuv

rep

mob

GmR

P102

P101

rbs-gfp

Omega TT

Pharmacia TT

Cla I (4046)

Pst I (3999)

Pme I (4020)

Sac I (3260)

Sal I (4032)

Spe I (3977)

EcoR I (3061)

EcoR I (3240)

Pac I (4012)

Pac I (4042)

BamH I (3439)

BamH I (3983)

Eco RV (4050)

Eco RV (4995)

Sma I (3991)

Sma I (4003)

Xba I (4025)

Xba I (4052)

102

4.3 Discussion

In this chapter the development of a promoter probe vector has been discussed. The

design of the plasmid has been in a modular form to facilitate the adaptation of the

vector as well as the incorporation of new reporters as they become available.

Initially the promoter probe was to be based on an adaptation of the IVET procedure,

in which selection against expression in the host occurs using SacB. However,

concerns about the toxicity of strong promoters in the environment led to the

construction of a fluorescent reporter system. To allow the development of a true

IVET vector, the cloning site PstI has been included in pOT1, along with flanking

SmaI sites. This allows either a negative selection marker, or a gene to complement

auxotrophy to be incorporated. The later would be a true IVET plasmid.

In the initial attempts to develop a promoter probe, the plasmid pPOT-K, pPOT-S,

pGOT-K and pGOT-S were constructed. There were several problems encountered

with these plasmids. The sensitivity of wildtype gfp was low. The pPOT vectors

could have been used to overcome this, as the sensitivity of PhoA is suitable for this

work. The disadvantage of this system however is the occurrence of native

phosphatases obscures results, especially in single cells. This could have been

overcome by using a phosphatase minus mutant but fluorescence of root material

would still have caused problems. However, a new version of GFP became

available, that was much brighter.

The stability of the pGOT vectors was tested in vivo and in vitro. In both cases,

stability problems were encountered. It appeared that deletions may be occurring

103

when the plasmid was in Rhizobium. This was probably due to recombination in the

Rec+ Rhizobium that was not occurring in Rec- E. coli. Due to this, a new promoter

probe vector was designed to overcome the problems.

The promoter probe pOT1 is based on the pBBR-1-MCS-5 plasmid. This is a small

broad host range plasmid with proven stability. It uses the Gentamycin resistance

gene making it suitable for use in Rhizobium. In pOT1 the gentamycin resistance

gene is transcribed in the opposite direction to gfpuv giving another safeguard against

unwanted transcription of gfpuv.

The promoter-less reporter gfpuv, incorporating a bacterial ribosome-binding site, is

utilised in pPOT1. The fluorescent protein allows us to monitor gene expression in

colonies and in single cells and its sensitivity is high. To protect gfpuv from

transcription from plasmid promoters and to prevent it from interfering with the

transcription of plasmid genes, two transcriptional terminators flank gfpuv. These

have different sequences to reduce the risk of deletions that prevented the use of the

pGOT vectors.

The plasmid pOT1 only has two transcriptional terminators, which have different

sequences. One Transcriptional terminator (Omega) prevents read into gfpuv and the

second (Pharmacia) prevents reading beyond gfpuv.

The modular construction of pOT1 allows other reporters to be incorporated (see

Table 4.1). As in our construction these could be cloned by PCR amplification using

a 5' primer with an identical 5' end to P69. This would incorporate a SpeI site and

104

ribosome-binding site. The 3' end of this primer would be complimentary to the new

reporter. The 3' primer should incorporate a SacI site.

Other reporter genes that could be considered for this vector include well studied

reporter systems such as lacZ, luxAB and gus. The bioluminescence of bacterial

luciferase encoded by luxAB (Reviewed by (Hill et al., 1993; Stewart & Williams,

1992)) has been used in a wide range of applications (Summary of papers (Stanley &

Stewart, 1990)). Its use in Rhizobium is also well established (Boivin & Chalifour,

1988; Paton et al., 1997). The gus A gene, encoding β-glucuronidase has also been

used in Rhizobium (Wilson et al., 1995) and is widely used as a reporter.

There is also a wide range of GFP derivatives available as reporters. These include

red shifted derivatives in which the excitation wavelength is increased. GFPmut1

has a 35-fold increase in fluorescence and has an excitation maxima at 488nm

(Cormack et al., 1996). The three derivatives (GFPmut1, mut2 and mut3)

constructed by Cormack et al. were optimised for use with FACS sorters. The

excitation and emission peaks are very close making them unsuitable for visual

analysis without using extensive filters.

Plasmid pPOT1 was subsequently used in the construction of a promoter library.

The characterisation of the plasmid and construction of the library is discussed in

chapter5.

105

Chapter 5

Construction of Promoter Libraries

5.1 Introduction

The previous chapter describes the construction of a plasmid designed to enable the

identification of environmentally induced promoters. Genomic DNA from R.

leguminosarum is cloned into the pOT1 vector to produce a promoter library.

Selection of Sub-libraries is also described, resulting in a non-expression library for

minimal media. During the selection of this library, a number of clones were

identified with differential expression on solid and liquid media. These were isolated

in another sub library. The construction and selection of these libraries is described.

5.2 Construction of library LB-1: Methods and Results

To construct a representative genomic library care must be taken at all stages to

minimise the effect of competition between clones. Several of the stages in

construction, for example the collection of transformants, are time consuming due to

the large numbers involved. These stages need to be split into batches that were

incubated at different times. It was essential to build into the protocol procedures to

compensate for the effect this could have on the overrepresentation of clones due to

different growth rates.

106

5.2.1 Cloning of 3841 genomic DNA into pOT1

In the original design of the pPOT and pGOT vectors a BamHI site was incorporated

into which Sau3A fragments containing promoters could be inserted in front of the

promoterless gfpuv. This procedure has the disadvantage that more than one

fragment can ligate in tandem to produce multiple inserts, adversely effecting the

representation of genomic DNA in the libraries and make mapping very difficult. To

prevent self-ligations the plasmid is de-phosphorylated with phosphatase. Complete

removal of the vector's 5' phosphate after digestion was too harsh resulting in the

plasmid ends being exonucleased and making further cloning impossible. For the

pOT1 vector a protocol was developed whereby multiple inserts were not possible

and the plasmid could not self-ligate. In this procedure genomic DNA was digested

with Sau3A and pOT1 with SalI. Both of these are six cutters, leaving four base

overhangs with the feature that the end two bases in these restriction sites are

complimentary (see Fig. 5.1). Partial polymerisation results in a two-base overhang,

which prevents self-ligation of either Sau3A or SalI termini but allows single

insertion events to occur.

The promoter library LB1 was constructed in E.coli genomic DNA from R.

leguminosarum 3841 was isolated (see methods 2.5.1.2) and partially digested with

Sau3A. Fragments above 2Kb were extracted from a 0.8% agarose gel using

Geneclean (Bio 101). In previous experiments, it had been noted that smaller

fragments were often retarded thereby biasing the pool for ligation. By using

fragments over 2Kb on the gel, it was anticipated that the bias would result in the

majority of inserts being between 1 and 2Kb. We hoped to reduce the number of

very small fragments in the library by extracting the DNA with Geneclean as it is

107

unsuitable for binding fragments below 0.5Kb. The single stranded ends of these

DNA fragments were partially in-filled using only dATP and dGTP leaving a 2 base

pair overhang, AG (Fig 5.1). Plasmid pOT1 was digested to completion with SalI

and its single stranded ends in-filled with dTTP and dCTP to leave an overhang of

CT. The genomic DNA partial fragments were ligated into the in-filled SalI site of

pOT-1

The ligated DNA was transformed into Supercompetent E. coli XL2 blue

(Stratagene) and plated on LA plates containing gentamycin (10 µg.ml-1).

Control ligations of the in-filled vector without insert DNA and transformations of

digested but non-ligated vector indicated that approximately 1% of the vector DNA

was undigested and a further 4.6% of the constructs in LB-1 were self-ligations with

no insert due to no polymerisation during in-filling.

Approximately 72,660 separate colonies were recovered from four ligation reactions

resulting in 22 separate transformation reactions. This was done to establish the

greatest genomic representation. Each transformation was plated and maintained as

a batch to control variation in growth. To maintain the growth of clones that may be

detrimentally effected in confluent growth on the plate, cells were spread on LA

plates at a dilution to obtain single colonies. The colonies from each plate were

washed into 5ml of LB+15% glycerol and the suspensions combined within each

batch.

108

SalI GFPuv

pOT-1 3841 Genomic DNA

Partial Sau3A Digest

SalI digest

nnnG TCGACnnn nnnCAGCT Gnnn

nnn GATCnnn nnnCTAG nnn

Polymerise in the presence of dTTP and dCTP

Polymerise in the presence of dATP and dGTP

nnnGTC TCGACnnn nnnCAGCT CTGnnn

nnnGA GATCnnn nnnCTAG AGnnn

Ligation

Fig 5.1: Construction of promoter library LB-1. See text for details.

Isolation of fragments over 2Kb

109

The batches were pooled together according to the ratio given below. To minimise

the effect of over representation of some clones within the final libraries the density

of bacteria after resuspension (measured as optical density (600nm)) of each batch

had to be taken into account:

V= [N / OD] x ________

[N / OD] min

Where V is the volume of the batch to be combined into the final library, x is the

batch number, N is the number of colonies in a batch, OD is the optical density of the

batch suspension at 600nm and min represents the batch with the minimum value for

N / OD.

This final cell suspension was stocked as LB-1

5.2.2 The distribution of genomic inserts in LB-1

To determine the representation of genomic DNA in the promoter library, LB-1, the

randomness of the insert sizes was investigated. A PCR screening procedure was

devised that would identify the insert sizes, thereby indicating the distribution of

fragment sizes and the number of clones with no insert (false off) in the final library.

A group of fifty-five random colonies from LB-1 were chosen and their DNA

amplified with primers P12 and P80 using the colony PCR method. These primers

amplify across the region containing the insert DNA and give a positive internal

control of vector DNA (0.8Kb) if there is no insert (Fig. 5.2).

110

A set of colonies was also checked before PCR for colony fluorescence and twelve

PCR fragments were obtained from fluorescent colonies and twelve from non-

fluorescent colonies. As expected all the fluorescent colonies gave PCR products

with insert DNA. Of the 12 non-fluorescent colonies, 9 had inserts and 3 had no

inserts. However, from the total PCR data, 7% of constructs did not have an insert.

These must all fall within the non-expression section of the library. Most of the

constructs with no insert are probably due to the incomplete digestion and infilling

encountered during cloning (section 5.2.1).

The distribution of insert sizes over fifty-one PCR reactions with inserts is shown in

Fig. 5.3. The mean insert size from this data set is 1.56Kb. The graph (Fig. 5.3)

indicates several interesting features. There appears to be two curves overlaying one

another, one with a peak between 0.01 and 0.5 and the second with a peak between

2.1 to 2.5. There is a predominance of fragments below 2Kb. Although attempts

were made to minimise very small inserts, there is a predominance of small

fragments. The observation that larger inserts, although present, are not prevalent

GFPuv TTTT

3841 genomic DNA

P12 P80

Fig 5.2: Diagram to show the location of primers P12 and P80, used in the PCR screen of LB-1, in relation to the insert DNA. TT indicates a transcriptional terminator.

0.8Kbp

111

indicates the factor of insert size biasing ligation events. The dramatic drop off of in

sizes over 2.6Kb is probably due to the preference for small fragments during

ligation. This high efficiency of small fragments to ligate also accounts for the peak

of inserts less than 0.5Kb. The mean insert size, 1.56Kb, however is acceptable for

this library. On the assumption that the average gene size is 1Kb, this mean insert

size would indicate that active promoters should be isolated on many fragments. It

should be noted that promoters are often active over whole operons and so it should

not be assumed that every gene has a promoter. The mean insert size also confirms

the assumption that extracting fragments over 2Kb would result in a predominance of

fragments below 2Kb.

02468

101214

Number of inserts in range

< 0.5 0.6 to1.0

1.1 to1.5

1.6 to2.0

2.1 to2.5

2.6 to3.0

3.1 to3.5

Insert size Kb

Fig 5.3 Distribution of Insert Sizes in LB-1 Obtained from PCR Amplification

112

5.3 Construction of the Promoter Library LB-2

The library LB-2 was developed by transferring LB-1 from E. coli into R.

leguminosarum strain 3841. Although consisting of the same clones as LB-1, this

library was given a new designation in Rhizobium. This reflects differences in

representation introduced during conjugation. It also allows this library to be

distinguished from subsequent sub libraries. The library consists of individual

clones, representative of the Rhizobium genome, in a Rhizobium host, stored frozen

in microtitre plates. The construction of LB-2 and the subsequent sub libraries is

summarised in Fig 5.4.

5.3.1 Construction of LB-2

Library LB-1 was conjugated into 3841 using the standard filter mating protocol with

the following modifications. To reduce the possibility of clonal propagation, the

donor, LB-1 was inoculated directly from frozen stock into LB and grown for 4.5 hr.

The conjugation was reduced to 9 hr to limit the number of replication cycles after

mating. These times had been previously determined as the minimum time to give

reasonable mating frequencies whilst minimising the number of siblings in the final

library (data not shown). The conjugation mix was stocked in glycerol (15%) at -

80°C and plated in batches on library selection plates (AMA supplemented with

glucose (10mM) ammonia (10mM) gentamycin (20 µg.ml-1) streptomycin (250

µg.ml-1) and nystatin (50 µg.ml-1) trimethoprim (10 µg.ml-1). This chemically

defined minimal medium was used to prevent induction of putative environmentally

induced promoters in the library. The antibiotics were used to maintain Rhizobium

(streptomycin) and the plasmid (gentamycin) and the nystatin was added to eliminate

113

Conjugation spread on AMA plates

Colonies picked into microtitre plates (AMS)

High fluorescent colonies patched on AMA

Borderline fluorescent colonies patched on AMA

Microtitre plates analysed using plate reader.

Microtitre plates frozen as LB-2

Low fluorescent colonies patched onto AMA

Patch plates screened using UV transilluminator

UV High fluorescent colonies patched in microtitre plates and frozen as LB-6

Borderline fluorescent colonies patched in microtitre plates and frozen as LB-5

After patching in microtitre plates borderline expression colonies washed of plates and bulked together as LB-4

High and borderline florescent colonies cut out of agar and the remaining colonies washed from plate and bulked together as LB-3

Fig 5.4 Construction of libraries LB-2 to LB-6

4 days growth at 27°C plus overnight incubation at 4°C

Primary screen

Secondary screen

114

fungal contamination. The trimethoprim was added to remove a minor contaminant

introduced during the stocking of LB-1. This was important as large numbers of

plates were being manipulated, incubated and stored for relatively long periods of

time and any loss due to contamination would have limited the representation of

clones.

To facilitate the future screening of the library, reference strains were selected from

the conjugation plates as representative of the following fluorescence levels seen on

minimal plates. RU1158 was selected as expressing high fluorescence levels on

agar, RU1160 gave no visible fluorescence and RU1159 gave a level of expression

that was just visible. As future analysis would involve identification of fluorescence

in single cells these were also tested under the microscope. The results confirmed

RU1158 and RU1160 as having high and no fluorescence respectively. RU1159 was

visibly fluorescent under the microscope however the excitation could be attenuated

to give a level that was just visible but which still allowed RU1158 to be highly

fluorescent.

Colonies (15,075) were individually picked from the conjugation plates (above) into

96 well microtitre plates containing 200 µl of AMS (supplemented with glucose

(10mM) ammonia (10mM) gentamycin (20 µg ml-1) streptomycin (250 µg ml-1)).

For a comprehensive library it is necessary to have complete representation of the

genomic DNA. For this an estimation of the number of colonies needed was made

using the following equation (Kaiser & Murray, 1985):

115

N= ln(1-p)/ln(1-x/y)

Where N is the number of constructs needed, p is the percentage chance of covering

all the genome, x is the average insert size in Kb and y is the genome size in Mb.

We calculated N using 99% for p and 1.56Kb for x (see section 6.2.2). As there was

no published size for 3841 genome, we estimated y using a value of 6.5Mb . This

was estimated from the genome size of Rhizobium meliloti 1021 (This consisted of

3450kb for the chromosome, 1340kb for pRme1021a and 1700kb pRme1021b

(Honeycutt et al., 1993)) and of R. leguminosarum bv. phaseoli (6435Kb) (Jumas-

Bilak et al., ). This indicated that 18943 inserts (1.6Kb) are needed to cover the

complete genome. However, as promoters operate unidirectionaly, this needs to be

doubled to approximately 38,000 inserts to ensure all promoters are included.

LB-2 contains a total of 15,075 individual colonies at mid log (OD630 over 0.15 (see

section 5.4.1)). This represents about 40% of the colonies needed to cover the

complete genome (see section 5.2.3). Time constraints within the project prevented

the isolation and screening of another 23,000 colonies necessary to give full genomic

representation.

The microtitre plates were incubated in a New Brunswick gyratory shaker in a

custom built holder at 125 rpm and 27°C. The holder consisted of an open topped

box, built of aluminium sheets, with two walls that could be adjusted and fixed to

hold the plates in place. After 3 days incubation the plates were screened in

preparation for the construction of sub-libraries (see section 5.4.1), and frozen in

116

15% glycerol. The screening consisted of measuring the OD630 and fluorescence of

the culture in the each well to obtain a relative fluorescence value, using a

Biolumin960 plate reader (Molecular Dynamics). This library, of individually

stocked Rhizobium clones was designated LB-2.

5.4 Construction of Sub-libraries

LB-2 is representative of both constitutive and inducible promoters. To produce a

promoter library that can be used to investigate environmentally regulated promoter

activity, a sub-library with strictly no constitutive promoters must be made. To do

this a screening protocol using a plate reader to accommodate the large numbers of

clones involved was developed. The range of promoter activities, and therefore

fluorescence meant that a simple on/off criterion for defining sub-libraries was not

possible. A threshold value for fluorescence needed to be determined below which

the promoter was deemed to be off. To quantify fluorescence, the number of cells

present within a well must be taken into consideration. A correction based upon

optical density was developed. As the microtitre plates were grown in batches, a

second correction was needed to account for growth differences. This was based on

an uninoculated well and the control strains RU1158, RU1159, RU1160 (see section

5.3.1) being inoculated on each plate. In this section these protocols and the

construction of the sub-libraries will be discussed

5.4.1 Calibration of plate reader

A Biolumin960 plate reader (Molecular Dynamics) was used to measure the large

number of fluorescence and optical density readings needed. To determine whether

117

it was appropriate to use this, several calibration experiments were necessary.

Within our laboratory, optical density readings to determine the cell density of

Rhizobium are carried out using a spectrophotometer at 600nm. The most suitable

filter set on the plate reader was at 630nm. The relationship between cell density and

optical density at 600nm and 630nm needed to be established.

It was also essential to determine if there was a linear relationship between optical

density and fluorescence, thus allowing us to determine the relative fluorescence

irrespective of the OD630. To allow maturation of GFP, only fluorescent values from

cultures grown to mid logarithmic phase of growth were analysed.

A suspension of LB-2 colonies was made in AMS and a two-fold dilution series

produced from the cell suspension. The OD600 of each dilution was measured using a

spectrophotometer. Aliquots of each dilution (200µl) were placed in a microtitre

plate. Each dilution was measured in triplicate and the results averaged. The OD630

and fluorescence values were measured using the plate reader.

5.4.1.1 The OD630 and OD600 correlate linearly to late logarithmic growth.

The OD600 and OD630 values show a good linear relationship, below OD600 0.7,

between the optical densities measured using these two methods (fig 5.5). The

optical density value for calculating the fluorescence of GFP was defined as an

OD630 of at least 0.15 as this represented a late log culture (approximately OD600

0.4), giving GFP time to mature.

118

5.4.1.2 Fluorescence is directly correlated to OD630

The fluorescence and OD630 values were plotted against the dilution of the culture

(fig 5.6). Fluorescence and OD630 have the same decay curve and are directly

proportional to the dilution. This confirmed that the fluorescence could be measured

over a wide range of OD630 and a correction made for optical density would give

directly comparable values for relative fluorescence.

Fig. 5.5 Relationship between optical densities measured using the spectophotometer and microtitre plate reader

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600

OD Spectophotometer (600nm)

OD

Pla

te re

ader

(630

nm)

Dotted line represents the defined threshold value for plate measurements. OD600 path length 1cm. Plate reader 200µl in flat bottom 96 well microtitre plate.

119

5.4.1.3 The threshold value for relative fluorescence.

To determine the threshold for relative fluorescence to be used as a definition of

clones that had promoter activity, a number of colonies isolated from the library LB-

2 were compared. The colonies displayed a range of relative fluorescence, V,

measured on the plate reader (V see section 5.4.2). These were also observed

microscopically for fluorescence and the threshold value for observable fluorescence

established. To aid the study of fluorescence levels, a series of isolates from LB-2

were identified with a range of fluorescence (Table 5.1). These were identified as

having GFPuv expression when grown in minimal broth, measured using the plate

reader. The fluorescence was then checked in colonies on the UV transilluminator

Fig. 5.6 Fluorescence is directly correlated with OD630 when measured using the plate reader

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20 25 30 35Dilution factor

OD

630

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

OD630 Fluorescence

Fluo

resc

ence

120

and under the microscope and a threshold value determined. This value was slightly

higher than the fluorescence for RU1159 as RU1159 was not always visibly

fluorescent. The value used in comparing relative fluorescence was defined as 1.25

fold the average value for RU1159 within a batch.

Strain Relative fluorescence (V)

RU1160 6,000 units

RU1159 17,000 units

RU1158 200,000 units

RU1311 2000 units

RU1312 10,000 units

RU1313 20,000 units

RU1314 30,000 units

RU1315 40,000 units

RU1316 62,000 units

RU1317 91,000 units

RU1318 199,000 units

RU1319 369,000 units

Table 5.1 The relative fluorescence (V) of the fluorescence standards isolated from LB-2. The first three strains are the original standards and are inoculated on all microtitre plates.

121

5.4.2 Screening of LB-2 using microtitre plate reader

The library, LB-2, was screened in microtitre plates for constitutive fluorescence

after growth in minimal broth, using a Biolumin960 plate reader. From the results of

this screening, sub-libraries were defined and constructed. Measurements of

fluorescence were made with a 405/10nm excitation filter and 505/10nm emission

filter. Optical density was measured at 630nm. Plates were analysed in batches of

28. This was a convenient number to incubate as it filled one layer in the incubator,

and a practical number to manipulate in one day, for example when hand picking

colonies into sub-libraries. The results for each well were analysed using an Excel

spreadsheet based on the following equation:

V=F-X / OD630-Y

Where V, the specific fluorescence is calculated from F the measured fluorescence

and OD630 the optical density for a particular well. The average fluorescence for the

blank uninoculated wells in the batch is given by x and the average OD630 for the

blank wells in the batch by Y. Wells with an OD below 0.15 were excluded from the

calculation.

The average value for strain RU1159 (Low fluorescent control) within a batch was

calculated and the threshold for fluorescence defined as 1.25 times this value (see

section 5.4.1). Schematic diagrams were produced from the spreadsheet indicating

wells with values below this threshold. The diagrams were actual size and could be

placed beneath the microtitre plate, indicating which colonies were to be patched

onto AMA plates.

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5.4.3 Selection of libraries LB-3, LB-4, LB-5 and LB-6

After screening using the plate reader, it was anticipated that a single sub-library

consisting of promoters deemed off in minimal broth would be constructed. A

second screening for fluorescence was carried out on minimal agar plates to ensure

the clones had not been mis-assigned. During this, it became apparent that a set of

clones had high fluorescence on the minimal agar plates. These were therefore

isolated, as were a set which were marginally fluorescent. In this way the off library

was completely silent in both broth and on plates. For this study, it was important to

produce a strictly silent sub-library however other sub-libraries were also stocked for

future studies.

To construct the sub-libraries, colonies below the fluorescence threshold were

patched from the microtitre plates (section 5.4.2) onto AMA plates (10mM glucose,

10mM ammonia, gentamycin (20 µg.ml-1) and streptomycin (250 µg.ml-1)). These

were incubated for 4 days and then at 4°C overnight to allow fluorescence to reach

its maximum level. The plates were re-screened using the UV transilluminater for

fluorescence. Colonies showing high levels of fluorescence were patched to fresh

plates and colonies with a borderline fluorescence level were patched onto separate

plates. The colonies that had been selected in this way were cut from the original

plates and discarded. Those colonies left (OFF colonies) were washed in to 5 ml

AMS+15% glycerol. This cell suspension (1ml)was stored at -80°C in 15% glycerol.

The cell suspension from each plate was checked for fluorescence using the

microscope and those with fluorescent colonies visible with attenuated excitation

were discarded. 100µl of the cell suspension from each plate was combined to

produce LB-3.

123

The plates containing high fluorescent and borderline fluorescent colonies were

incubated and patched into microtitre plates. The microtitre plates were incubated

and glycerol (to 15%) added to the wells before storing the clones at -20°C as LB-5

(Borderline fluorescence) and LB-6 (High fluorescence on plates). The borderline

and high fluorescent colonies were pooled from the plates in 15% glycerol and stored

in individual tubes at -80°C. The cell suspension for the borderline colonies was

combined as above and designated LB-4 (Fig 5.4). The final composition of the

libraries is given in Fig 5.7.

5.5 Testing of the library LB-3

To establish if constructs in the library LB-3 were inducible under variable

conditions, 20,000 colony-forming units were plated on 200 AMA plates. These

plates were phosphate limited (20µM) and had hesperitin (1µM) added. The carbon

source was succinate (10mM) rather than glucose. Colonies that were fluorescent

Fig 5.7 Composition of Promoter library LB-2(Number is total colonies).

7%

71%

1% (128)4% (558)

17% (2549)

78%(11841)

constitutive expression in AMSExpression below threshold in AMS But high expression on AMA plates (LB-6)Expression below threshold in AMS with borderline expression on AMA plates (LB-5)Constructs estimated not to contain an insert (see section 6.2.2) (LB-3)Expression below threshold in AMS and on AMA plates containing inserts (LB-3)

Note: LB-3 contains a subset of constructs with no inserts. This was estimated as 7% of the total colonies (see section 6.2.2)

124

under these conditions were screened on the three inducing conditions (Phosphate

limitation, hesperitin induction and succinate) individually. Nine phosphate

limitation induced colonies, one succinate induced, two hesperitin induced and one

hesperitin and succinate induced colonies were identified (Table 5.2), indicating that

library clones could be induced by altered growth conditions.

Isolate Details

RU1234 Induced by phosphate limitation


RU1236 Induced by succinate and hesperitin




RU1245 Induced by hesperitin




RU1249 Induced by hesperitin


RU1251 Induced by succinate

Table 5.2 Summary of the LB-3 library isolates (pOT1) induced by growth in limited phosphate, succinate and hesperitin.

125

5.6 Discussion

In this chapter the construction of a series of promoter libraries was described. The

calibration of the plate reader supports the basis of the protocol as satisfactory for the

selection of a promoter sub-library in which the clones are non-fluorescent when

grown under minimal defined conditions. Within this library (LB-3) there are a

proportion of clones with no inserts. PCR data indicated two values for this. When

the library LB-2, containing all promoter clones, was tested 7 % of the constructs

contained no insert DNA. However, when twelve non-fluorescent clones were

tested, 25% of then contained no insert. This discrepancy is in part accounted for by

the selection of clones. If no selection for fluorescence is made, the number lacking

inserts is lower as the population being tested contains clones that are fluorescent and

therefore must have an insert. Secondly the sample size when the non-fluorescence

colonies were tested is small allowing for the possibility of further discrepancies.

The implications of these clones containing no insert would be to necessitate the

production of a larger library. However, time constraints prevented a complete

library being constructed.

The PCR results also indicated a bias in the distribution of insert sizes. There were

two peaks, one below 0.5Kb and the second between 2.1 and 2.5Kb. These were due

to the different ligation efficiencies biasing smaller fragments and the method used to

isolate the fragments to reduce the number of small fragments.

The sub library LB-3 was defined as those clones that had fluorescence below a

defined threshold in minimal broth and also no observable fluorescence on minimal

plates. This represented 78% of the complete promoter library, LB-2. It should be

126

noted that approximately 50% of the complete library would be expected to be non-

fluorescent as promoters operate unidirectional and the cloning of insert DNA was

random.

A sub library, consisting of clones that had marginal expression during the second

screening on plates, was constructed. This was in effect a safety buffer to allow the

expression of clones in LB-3 to be strictly off on minimal conditions. It can be

screened for environmentally expressed promoters in the same way as LB-3 however

care needs to be made of attenuation of the excitation light. This library was shown

to be inducible by growth on phosphate limited media and by hesperitin and

succinate.

The sub-library LB-6 consisted of clones that were non-fluorescent in broth but were

significantly fluorescent on plates. It was possible that this group contains promoters

that are regulated by the physical state of the media. This could include cell-cell

communication genes and genes specifically needed to grow on solid medium.

Further characterisation of these clones was carried out and is discussed in chapter 6.

The library LB-3 was used to identify environmentally induced promoters of R.

leguminosarum, during the infection of roots. This is discussed in chapter 7.

127

Chapter 6

Putative Media Regulated Promoters

6.1 Introduction

During the construction of the promoter libraries, the sub library LB6 was created

and stored at -20°c in microtitre plates. This library contains pOT1 plasmids that

have low fluorescence when examined in microtitre plates but had visible

fluorescence when checked on agar plates. In this chapter we discuss the further

screening of these isolates. In particular this gave an indication of whether the

library contains inducible promoters that could be detected under different

conditions. This group of isolates was also interesting in its own right as their

selection indicated that they contain promoters that are regulated by the physical

state of the medium they are grown on.

6.2 Screening of library LB-6: Methods and Results

During the construction of library LB-6, only 92 isolates from about 10,000 colonies

were retained. These were stored at -20°C in a microtitre plate (200µl AMS, 15%

glycerol). To examine expression of GFP in these clones, a fresh microtitre plate

(200µl AMS, 10mM glucose, 10mM Ammonia per well) was inoculated from the

stored plate and grown for 3 days. The isolates were then replica plated into a fresh

microtitre plate and onto AMA plates (10mM glucose, 10mM ammonia) and

incubated for a further 3 days.

128

Fluorescence measurements were taken for both the agar plates and the liquid culture

from the microtitre plate as follows. The samples grown in the microtitre plate were

mixed by pipetting and 50µl transferred into 150 µl of AMS in a fresh microtitre

plate. This was done to ensure all samples were 200ml and therefore had the same

path length for the plate reader. The samples grown on agar plates were prepared by

transferring a piece of the colony to 200 µl of AMS in a microtitre plate. Pipetting

repeatedly through a Gilson tip dispersed the colony. The fluorescence value and

OD630 were measured for the plates and for each sample V was calculated (see

section 5.4.1). Isolates were considered to be significant if the ratio of V from agar

growth to V from broth growth was greater than 3. This is designated experiment 1

(Ex1, Table 6.1).

Of the original 92 isolates 24 fulfilled this criteria (see Table 6.1:Ex1). These

isolates were colony purified and given formal strain designations. They were

gridded in a microtitre plate, grown for three days and then replica plated onto fresh

microtitre plates and onto AMA plates. After 3 days growth in liquid and solid

media the fluorescence was measured as before. The results are designated

experiment 2 (Ex2).

When the purified isolates were plated to single colonies and observed using the UV

transilluminator, fluorescence was not always consistent throughout the colonies

(See section 6.2.1). These distributions need to be considered carefully when

interpreting the numeric data from the plate reader, as it is apparent that a widely

different value for the agar grown colonies could be obtained depending on what part

of the colony was resuspended.

129

As some of the fluorescence on plates occurred only in the original streak line, we

wanted to establish if any promoters were being expressed in late stationary phase.

Rhizobium is fully grown in about 2 days in the microtitre plates, therefore 3 day

cultures should be well into stationary phase. However, it was important to test

whether further incubation might alter gene expression further. We also wanted to

know if the effect of the solid surface was due to the agar or the presence of any solid

surface for growth. To investigate this, the isolates were grown in universals (10ml

AMS, 10mM glucose, 10mM ammonia) and streaked on AMA plates (10mM

glucose, 10mM ammonia) solidified with agar, agarose (2%) or gelatine (20%).

Samples were taken after 4,7 and 10 days growth and V calculated as before. This

has been designated experiment 3 (Ex3, Table 6.1).

The results for the plate readings are summarised in Table 6.1. The ratio of

fluorescence in colonies over fluorescence in liquid was determined three times

independently. In the first experiment the original isolate was used and the growth

time was 3 days. In the second experiment a colony-purified isolate of the original

isolate was grown for three days. In the final experiment, the colony-purified isolate

was used, however, the fluorescence ratio was determined after 10 days of growth

with the fluorescence on plates observed on the transilluminator at 4 and 7 days.

This allowed us to determine if any isolates had altered expression in late stationary

phase. After the three experiments, 24 samples showed higher specific fluorescence

on solid media compared to growth in liquid media. It was beyond the scope of this

project to examine all of these exhaustively, however, a sample was sequenced. All

the samples were grown to late stationary phase and most showed no change in

130

expression after 4 days. However, the expression of strains RU1216 and RU1188

increased significantly during late stationary phase, whereas the other strains were

constant during this period. This indicates that these may contain promoters active

during extreme starvation.

Table 6.1 Genes Expressed at higher levels on plates compared to liquid culture

Strain Plasmid Solid/Liquid Product

(Expression Level and DNA Sequence) Ex1 Ex2 Ex3 RU1197 pRU509 3.4 1.6 1.5 Uniform high RU1198 pRU511 5.3 2.6 5.6 Single colonies bright and streak tips RU1199 pRU512 6 0.8 1.4 Uniform high RU1201 pRU514 3.4 1.3 3.3 Uniform high RU1202 pRU515 3.1 1.5 1.3 Uniform high RU1203 pRU517 4.5 N/d 2.6 Uniform high RU1204 pRU518 3.2 0.5 2 Extreme papilation RU1182 pRU496 36 0.98 1.7 Uniform low. No Database hits RU1207 pRU521 4.2 1.9 1.9 Uniform high. RU1253 pRU523 4.8 2.4 1.9 Inverse fisheye RU1209 pRU524 4.3 0.7 2.5 Inverse fisheye RU1257 pRU525 7.4 4.4 1.6 Uniform low RU1212 pRU526 3.9 1.6 3.5 Not analysed RU1214 pRU527 4.1 0.9 5.1 Low on, mild papilation RU1215 pRU528 3.1 8.8 5.6 uniform low RU1216 pRU529 3.7 2.5 1.9 Uniform medium. Phytochrome 34% over 70

residues, then 34% over 41 slightly further along.

RU1256 pRU530 4.3 3.7 5 Single colonies on and at the tips of streak lines. Absolute expression in liquid 3.8 fold higher on day 10 compared to day 4.

RU1220 pRU531 3.7 2.1 3.3 uniform on RU1183 pRU499 inf 4.3 7.8 fixND ie component of CBB3 cytochrome

oxidase complex, only on in single colonies. RU1256 pRU534 12.5 2.1 1.7 Uniform on. RU1226 pRU536 3.5 2.4 4 Uniform on. RU1188 pRU498 6 5.7 1.4 Very bright but in tight focal points. metX i.e.

homoserine O-acetyltransferase (43% identity over 67 residues), Absolute expression in liquid 5.2 fold higher on day 10 compared to day 4.

RU1184 pRU500 33 3.2 0.8 Mainly on in single colonies and outside streaklines. 16S rRNA dimethyltransferase 40% identity over 49 residues.

RU1230 pRU539 5.4 2.3 2.3 Not analysed. Notes: Plasmids that have been partially sequenced are indicated in bold. N/d ~ not determined Inf ~ Infinity Uniform ~ all colonies on plate have uniform fluorescence.

131

6.2.1 Plate Observations

When observing the isolates grown on plates some interesting distributions of

fluorescence within the colonies were noted. Some of the colonies grew with

fluorescent papilation and others had fish eye distributions where the centre of the

colony was fluorescent. This indicates that some of the promoters are being

triggered by subtle changes within the colony. This observation was also important

in deciding the significance of the results of fluorescence obtained from the plates.

The comparison of expression on plates solidified with agar and agarose showed no

significant difference in expression values. Strains grown on gelatine produced flat,

very small, undefined colonies and were not analysed further.

6.2.2 Sequence data

Strains RU1182, RU1216, RU1183, RU1188 and RU1184 were sequenced from the

pOT reverse primer and the sequence data subjected to a BLAST search. The

computation was performed at the ISREC using the BLAST network service. The

sequence data is shown in Fig. 6.2 along with the BLAST results.

132

RU1182 (pRU496)

1 TTAACTYAMA CGCTTTAACT ATAGMCTGAT ATGAMCCCGA TTYTGAMTGA 51 TGCRGTTGYY MTGATCMCAT GAGATTTGAC ATGAACTAGT GWCATCHCAG 101 TGAMAVTGAA AGTGACACGG AWAGWGWCYC GRCACAAACG AAACTCWTTG 151 WCAGTGAGAa ACTGAGAMAM TAWAAGWGAT CATBTGAATA AHKGRGACCT 201 CTTTGWMAGA TAAATAASTC AGGTCMCaCT ATAAVTVTGA dATCTCkGtG 251 ACCKGYCTCT GAkATRATAT CTCTMTATMT GATAGSTMTC TGATATGTGA 301 TATGCTGATA TGCTGSTCCG GTGRTMMTHK KACCGTTTta cMTRAKGRMM 351 GTTCcYCCTG GTCTCTCTYY GATGATGRTK TTCTCKGAAG AGgTATGYTG 401 wgTTGTCCGG GGAaATTATA T

BLAST search gave no hits for this sequence.

____________________________________________________________________

RU1216 (pRU529)

1 AGGTTTAAAC AGTCGACTCT AGACTTAATT AAAGTTTAAA CTCTAGATGT 51 CGATCAGTGT GACGCGGATG GTTTCGGCGA CACGGTGCTC AGCCTCCGTG 101 AAGGGGAGAG AGCGACCACG CACCAATTCC GACCACGCCT CGAAACTCTT 151 GCGCGGCGTC AGSGGGACCM GTTCGGGCCG TATTCCACCG GCTTGTGCGG

Fig. 6.2 Sequence data and BLAST results for strains RU1182, RU1216, RU1183, RU1188 and RU1184. For the Blast results, the name of the gene and organism from which it was isolated are shown in bold. The database reference number is shown in bold and underlined. Ambiguity codes for DNA sequence: Code Unresolved bases M AC R AG W AT S CG Y CT K GT V ACG H ACT D AGT B CGT N ACGT

133

201 GTCGCCGCCC CAGCGGACGG TGCGCACCAA TTCCTGACGG AAAAGCACGA 251 CATAATCGCG CGGCGAACGT GAGATCGGGA TGGCGAGCAT GCCTGCCACA 301 GCATCGTCGA CCTCAAGATC CGGATAGGCT TCCGCGAGCC TATCCACGGC 351 ATAGATGCGG CCGGCGGCGT TGCGGTTGAG ATGGCGGACG AGAGCTGCGA 401 ACCTCTCTCA TTCGGCCCGA TGCCGGMAAG AGCCAGCCGG CCGTTGATCc 451 AGACGCCGAT CCcSGTGGGA GGAATGGCGT CGGCGAVCGC TTCGATCAGC 501 CAGGCCGGWT CGTCGAGMMG GGTCSMHTTG TCBmAMGGAG GTAAARCSGT 551 CGCGTRCGxG SGYYMAXCCM mAMMAGTGMS GRCRCCSGAC GSCGCGRGGT 601 GTGHTGGGCG CRCVCGMRCG CATYACACGY GCGCGCRTGT GKCVACVTGY 651 GCGACTDYXX RTACASASDV TATCRCGAVR CATACACGMW CGTGCACWSm 701 GHAYACASAC RCAYGTSMTV KATMACAYMc ATCTCDGAVY GCRTMGMGCG 751 CKCTACWGBG SACGCKHHDC ACWTGMHAGT GTGTVTGTBB XGVGCGBGCA 801 GCAKMGMCGC MSCRCATG

sp|Q55168|Y473_SYNY3 HYPOTHETICAL 84.2 KD PROTEIN SLR0473 gi|1001165|gnl|PID|d1010958 (D64001) phytochrome [Synechocystis sp.] Length = 748 Score = 48.8 bits (114), Expect = 1e-09 Identities = 24/70 (34%), Positives = 42/70 (59%), Gaps = 1/70 (1%) Query: 392 LVRHLNRNAAGRIYAVDRLAEAYPD-LEVDDAVAGMLAIPISRSPRDYVVLFRQELVRTV 216 L++ L ++ L++ YPD + +G+LAIPI+R ++++ FR E+++TV Sbjct: 391 LLQWLENREVQDVFFTSSLSQIYPDAVNFKSVASGLLAIPIAR--HNFLLWFRPEVLQTV 448 Query: 215 RWGGDPHKPVE 183 WGGDP+ E Sbjct: 449 NWGGDPNHAYE 459 Score = 35.2 bits (79), Expect = 1e-09 Identities = 14/41 (34%), Positives = 27/41 (65%) Query: 168 VPLTPRKSFEAWSELVRGRSLPFTEAEHRVAETIRVTLIDI 46 + L PR+SF+ W E+VR +SLP+ E + A ++ ++++ Sbjct: 467 IELHPRQSFDLWKEIVRLQSLPWQSVEIQSALALKKAIVNL 507 gi|1800219 (U56731) phytochrome C [Sorghum bicolor] Length = 1135 Score = 40.6 bits (93), Expect = 2e-05 Identities = 23/60 (38%), Positives = 34/60 (56%), Gaps = 2/60 (3%)

134

Query: 350 AVDRLAEA-YPDLE-VDDAVAGMLAIPISRSPRDYVVLFRQELVRTVRWGGDPHKPVEYG 177 + D L EA YP + + V GM AI IS +D++ FR + ++WGG H+PV+ Sbjct: 483 STDSLVEAGYPGAAALREVVCGMAAIKISS--KDFIFWFRSHTTKEIKWGGAKHEPVDAD 540 Query: 176 PN 171 N Sbjct: 541 DN 542 Score = 29.0 bits (63), Expect = 2e-05 Identities = 12/34 (35%), Positives = 23/34 (67%) Query: 156 PRKSFEAWSELVRGRSLPFTEAEHRVAETIRVTL 55 PR SF+A+ E+V+ RS+P+ + E ++++ L Sbjct: 548 PRSSFKAFLEVVKWRSVPWEDVEMDAIHSLQLIL 581 sp|Q39557|PHY2_CERPU PHYTOCHROME 2 gi|1314837 (U56698) phytochrome photoreceptor CERPU;PHY0;2 [Ceratodon purpureus] Length = 1121 Score = 39.5 bits (90), Expect = 2e-05 Identities = 23/56 (41%), Positives = 32/56 (57%), Gaps = 2/56 (3%) Query: 350 AVDRLAEA-YPDLEV-DDAVAGMLAIPISRSPRDYVVLFRQELVRTVRWGGDPHKPVE 183 + D LA+A YP + DAV GM A I+ +D++ FR + V+WGG H P E Sbjct: 477 STDSLADANYPGAHLLGDAVCGMAAAKITA--KDFLFWFRSHTAKEVKWGGAKHDPAE 532 Score = 30.1 bits (66), Expect = 2e-05 Identities = 13/34 (38%), Positives = 23/34 (67%) Query: 156 PRKSFEAWSELVRGRSLPFTEAEHRVAETIRVTL 55 PR SF+A+ E+V+ RSLP+ + E ++++ L Sbjct: 541 PRSSFKAFLEVVKRRSLPWEDVEMDAIHSLQLIL 574 pir||S58130 phytochrome - moss (Ceratodon purpureus) (fragment) Length = 577 Score = 39.5 bits (90), Expect = 2e-05 Identities = 23/56 (41%), Positives = 32/56 (57%), Gaps = 2/56 (3%) Query: 350 AVDRLAEA-YPDLEV-DDAVAGMLAIPISRSPRDYVVLFRQELVRTVRWGGDPHKPVE 183 + D LA+A YP + DAV GM A I+ +D++ FR + V+WGG H P E Sbjct: 477 STDSLADANYPGAHLLGDAVCGMAAAKITA--KDFLFWFRSHTAKEVKWGGAKHDPAE 532 Score = 30.1 bits (66), Expect = 2e-05 Identities = 13/34 (38%), Positives = 23/34 (67%) Query: 156 PRKSFEAWSELVRGRSLPFTEAEHRVAETIRVTL 55 PR SF+A+ E+V+ RSLP+ + E ++++ L

135

Sbjct: 541 PRSSFKAFLEVVKRRSLPWEDVEMDAIHSLQLIL 574

____________________________________________________________________

RU1183 (pRU499)

1 TCGTCGAAAT AGCCTGAHAT ATCGACTTTC CGCCGTTAGC CTGCCGGCGA 51 AAAATCAACG TTCCTGACGA GCAGGGCAGC CCCCACCGCG AGGCAGAAGC 101 AAAGTATTCC CATATGGACC GCAAAGAGAT GATCATGTGC GAATGCVGCT 151 CCTAGCAMCG CTAGAAACGC CGCGACCGCG ATCACCATCG TTTCCGTTGT 201 ATAATTCATG ATGTCGTCCC CAGTGCGCCG ACGACCGCGT CGCGGCCAGC 251 TTTCTCGCCA GGACTGTCAC GAAGCCGCGA ATTGCTCCTT GATCTACATC 301 AACAAGAGGC CAGCAAGACG GGGCGAACCG CCGCGGTTAC AAACTGTTAC 351 AAACTTTCAC CGGTTAGCAC TGTCCGATCA TTCCCGCTGT GACGCAGAGG 401 CGTTCCAATT GTGCCGACCG ACAATGAGGA ACACGACGAT GCTGATGCAG 451 AAMACCcAGG KGTTCRAACA TGCCGAAAAT KRACGACCRg BCRTWYGMAC 501 CGGKYCMAAT CCGKCARAGH TTTCCAMAAH TGGGGGgRGs MWXTTCbCSG 551 SSRAAxCCAA CGGGGGAGGG CMARGG

trembl|P72288 (FIXND) FIXND (EC 1.9.3.1). [RHIZOBIUM LEGUMINOSARUM] Length = 540 Minus Strand HSPs: Score = 310 (109.1 bits), Expect = 1.3e-26, P = 1.3e-26 Identities = 61/69 (88%), Positives = 63/69 (91%), Frame = -2 Query: 209 MNYTTETMVIAVAAFLAXLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSPAG*RR 30 MNYTTETMVIAVAAFLA L AAFAHDHLFAVHMGILCFCLA GAA+L+R VDFSPAG RR Sbjct: 1 MNYTTETMVIAVAAFLALLLAAFAHDHLFAVHMGILCFCLAAGAAVLIRRVDFSPAGQRR 60 Query: 29 KVDXSGYFD 3 KVD SGYFD Sbjct: 61 KVDISGYFD 69 trembl|O06656 CBB3-TYPE CYTOCHROME OXIDASE COMPONENT FIXN (EC 1.9.3.1). [RHIZOBIUM LEGUMINOSARUM] Length = 540 Minus Strand HSPs: Score = 291 (102.4 bits), Expect = 1.7e-24, P = 1.7e-24 Identities = 57/69 (82%), Positives = 60/69 (86%), Frame = -2

136

Query: 209 MNYTTETMVIAVAAFLAXLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSPAG*RR 30 MNYTTETMVIAVAAFLA L AAFAHDHLFAVHMGILC CL +GA L+VR VDFSPAG +R Sbjct: 1 MNYTTETMVIAVAAFLALLVAAFAHDHLFAVHMGILCLCLVMGAVLMVRKVDFSPAGQQR 60 Query: 29 KVDXSGYFD 3 VD SGYFD Sbjct: 61 NVDRSGYFD 69 trembl|Q52826 (FIXN) FIXN (FRAGMENT). [RHIZOBIUM LEGUMINOSARUM] Length = 187 Minus Strand HSPs: Score = 275 (96.8 bits), Expect = 1.9e-23, P = 1.9e-23 Identities = 54/55 (98%), Positives = 54/55 (98%), Frame = -2 Query: 209 MNYTTETMVIAVAAFLAXLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSP 45 MNYTTETMVIAVAAFLA LGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSP Sbjct: 1 MNYTTETMVIAVAAFLALLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSP 55 trembl|P72284 (FIXNC) FIXNC (EC 1.9.3.1). [RHIZOBIUM LEGUMINOSARUM] Length = 539 Minus Strand HSPs: Score = 275 (96.8 bits), Expect = 9.5e-23, P = 9.5e-23 Identities = 54/55 (98%), Positives = 54/55 (98%), Frame = -2 Query: 209 MNYTTETMVIAVAAFLAXLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSP 45 MNYTTETMVIAVAAFLA LGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSP Sbjct: 1 MNYTTETMVIAVAAFLALLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSP 55 trembl|P95600 (FIXN) FIXN (EC 1.9.3.1). [RHIZOBIUM ETLI] Length = 540 Minus Strand HSPs: Score = 270 (95.0 bits), Expect = 3.4e-22, P = 3.4e-22 Identities = 52/69 (75%), Positives = 56/69 (81%), Frame = -2 Query: 209 MNYTTETMVIAVAAFLAXLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSPAG*RR 30 MNYTTETMVIAVAAFLA L AAFAHDHLFAVHMGILC CL G LLVRN +FSP G +R Sbjct: 1 MNYTTETMVIAVAAFLALLAAAFAHDHLFAVHMGILCLCLVAGTLLLVRNAEFSPTGQQR 60 Query: 29 KVDXSGYFD 3 K + +GY D Sbjct: 61 KTELTGYCD 69 sp|P98055|FIXN_AGRTU (FIXN) CYTOCHROME C OXIDASE POLYPEPTIDE I HOMOLOG (EC1.9.3.1). Length = 539 Minus Strand HSPs: Score = 153 (53.9 bits), Expect = 1.6e-09, P = 1.6e-09 Identities = 36/69 (52%), Positives = 40/69 (57%), Frame = -2

137

Query: 209 MNYTTETMVIAVAAFLAXLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSPAG*RR 30 MNYT ET A+ AF A LGAAFAHD LFA HM +L F L V LL+R V F P Sbjct: 1 MNYTLETADRALGAFPALLGAAFAHDSLFAAHMWVLFFTLVVSTLLLLRRVSFLPPVAGP 60 Query: 29 KVDXSGYFD 3 + YFD Sbjct: 61 PCRRTEYFD 69 sp|Q05572|FIXN_RHIME (FIXN) CYTOCHROME C OXIDASE POLYPEPTIDE I HOMOLOG, BACTEROID (EC 1.9.3.1) (CYTOCHROME CBB3 SUBUNIT 1) (HEME B/COPPER CYTOCHROME C OXIDASE SUBUNIT). Length = 539 Minus Strand HSPs: Score = 143 (50.3 bits), Expect = 1.9e-08, P = 1.9e-08 Identities = 28/56 (50%), Positives = 36/56 (64%), Frame = -2 Query: 209 MNYTTETMVIAVAAFLAXLGAAFAHDHLFAVHMGILCFCLAVGAALLVRNVDFSPA 42 M +T E +V++V AFLA +GA A D LF HM +L F L G +L+R VDF PA Sbjct: 1 MKHTVEMVVLSVGAFLALVGAGLAQDRLFGAHMWVLFFALLAGTLVLMRRVDFRPA 56

____________________________________________________________________

RU1188 (pRU498)

1 TCCCAATAGC CAGCRCTTAC ATCCGCCTCC TTATATTTTC CCGCCGCGTG 51 ACTGTWTCCG CTGAAATAAT GCGGAATCAG GATGGCATTA TCCTTGGCGT 101 CGTTCAGCGT GCCATAAGTT TCATACCCCA GCCGCATTTC GGGAACCGTC 151 CTGCCTCCAC GTGTTACGAA GTTTTTCAGG GTAAATTCCC GCTTTTCAAC 201 CAATGGCTCG TACGCCAATG CAGGGCCGGC CATCATGGCA ATAGTGATGA 251 GTATTTTCAG ACGCATTTAC TTCTCCTTGG TCGGATTACC AACGAAACAA 301 AGCGGTGGCC TCAGCCTTCC AAAGCCGTAT CCTAGTCACT GGATTTCACA 351 TTGATCCAGG TCAAAACTTC TCAGCTTCTA AGGGGCTTAT TCGCCCCTGT 401 GGGATCATKT CAGCCGCCAG ACCGCGGCGA CTTCTCAAGA GTAATACTCT 451 GGKGCCTTCT TGRRGGGYGA MCACGSACTG GGTCBGTTAC SGGGTTHCBC 501 GTTTTYCCMC GTCCTTtCCG CGTTTKTTCG CmXCTTYTKT WCAGGCGAaM 551 GTTYMMCvBY GRGVRGRKAA MCVCTCGAA

trembl|P94891 (METX) HOMOSERINE O-ACETYLTRANSFERASE. [LEPTOSPIRA MEYERI]

138

Length = 379 Minus Strand HSPs: Score = 157 (55.3 bits), Expect = 3.1e-10, P = 3.1e-10 Identities = 29/67 (43%), Positives = 41/67 (61%), Frame = -2 Query: 203 LVEKREFTLKNFVTRGGRTVPEMRLGYETYGTLNDAKDNAILIPHYFSGXSHAAGKYKEA 24 +V + ++ GG T+ + + YETYGTLN+ KDNAIL+ H SG +HAAG + E Sbjct: 16 VVYTQSIRFESLTLEGGETITPLEIAYETYGTLNEKKDNAILVCHALSGDAHAAG-FHEG 74 Query: 23 DVSAGYW 3 D G+W Sbjct: 75 DKRPGWW 81 sp|Q10341|YD9D_SCHPO (SPAC19G10.13) HYPOTHETICAL 56.4 KD PROTEIN C19G10.13 IN CHROMOSOME I. Length = 504 Minus Strand HSPs: Score = 107 (37.7 bits), Expect = 0.0029, P = 0.0029 Identities = 27/79 (34%), Positives = 43/79 (54%), Frame = -2 Query: 239 AMMAGPALAYEPLVE--KREFTLKNFVTRGGRTVPEMRLGYETYGTLNDAKDNAILIPHY 66 ++ +GP Y +V K+ + K F+ G +P+ + YET+GTLN NAIL+ Sbjct: 65 SLSSGPEPVYGKIVSGFKKFYHNKPFLCDHGGILPKFEIAYETWGTLNKDHSNAILLHTG 124 Query: 65 FSGXSHAAGKYKEADVSAGYW 3 S SHA + E + + G+W Sbjct: 125 LSASSHAHS-HPE-NTAPGWW 143 trembl|O13389 (CYSC) PUTATIVE HOMOSERINE O-ACETYLTRANSFERASE. [EMERICELLA NIDULANS] Length = 525 Minus Strand HSPs: Score = 107 (37.7 bits), Expect = 0.0031, P = 0.0031 Identities = 27/84 (32%), Positives = 43/84 (51%), Frame = -2 Query: 254 ILITIAMMAGPALAYEPLVEKREFTLKNFVTRGGRTVPEMRLGYETYGTLNDAKDNAILI 75 +L ++ +GP +Y +R + + + G +PE + YET+G LN+ KDN IL+ Sbjct: 79 LLSARSLGSGPEPSYTAGHHERFHSDEPLLLDWGGLLPEFDIAYETWGQLNEKKDNVILL 138 Query: 74 PHYFSGXSHAAGKYKEADVSAGYW 3 S SHA EA+ G+W Sbjct: 139 HTGLSASSHAHST--EANPKPGWW 160 sp|P15223|SCX1_CENNO TOXIN 1 (TOXIN II.14). Length = 65 Plus Strand HSPs: Score = 67 (23.6 bits), Expect = 0.52, P = 0.40 Identities = 18/57 (31%), Positives = 26/57 (45%), Frame = +3 Query: 162 CYEVFQGKFPLFNQWLVRQCRAGHHGNSDEYFQTHLLLLGRITNETKRWPQPSKAVS 332 CY++ GK N + R+CR H G S Y +++ T WP P+K S Sbjct: 15 CYKL--GK----NDYCNRECRMKHRGGSYGYCYGFGCYCEGLSDSTPTWPLPNKTCS 65

139

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RU1184 (pRU500)

1 CTTGCCGCAG GAXAGCTCTT GGACGACCTC GGCAAGTTGC GGGAXACCAT 51 ACCCCGGCTT GAAAAACATG CCTTGATAAA TATCAAAGGA AATAGCTTGC 101 CGAATGAATG GGGAAGGTAC GGHAGGGGAG CTACAAGAGC CGCAGCAGCC 151 TCGGATCAGG GCACGGATTA TCGAACGTGA CGGCCCAGGW ACAGATAGGC 201 GGTGGGATAC AGCGGCATGC AAATCACTTG CTTGGTGAAT AGCGGATCGA 251 CGGTTCGCAT CCGTTCAAGA GCACTAAACG GGCGGGGCCC CGGCTGAKTC 301 GTGCCGATTT GCAACGATTG CTTCGAGTCT TTCTCGCAAC ACCTGAGCGG 351 GAACTGTAAT CTGGGACGGK WTTKGGGCTG GGATTAGAAC GTTTCCATTT 401 ASGKTATCGG GGGGACTGSG XAGGSTTTTG SGCGDGBGGG DCTGTTTGgW 451 GAtGTSYGCK CYVYGTTTTC CWKARGCCGG GTTTWGTTGT CGSGGTTTTG 501 TtSCCGSyYG GGCGKGGRAA AaGGKWCCcA SCGTAGGWCC GGDWWTTGCC 551 GWAGGGTTGG GKKGAGgRCS

trembl|O25972 (HP1431) 16S RRNA (ADENOSINE-N6,N6-)-DIMETHYLTRANSFERASE (KSGA). [HELICOBACTER PYLORI] Length = 271 Minus Strand HSPs: Score = 75 (26.4 bits), Expect = 9.0, P = 1.0 Identities = 20/49 (40%), Positives = 28/49 (57%), Frame = -2 Query: 161 P*SEAAAALVAPLPYLPHSFGKLFPLI--FIKACFSS-RGMVSRNLPRS 24 P E A A +A P+ + K F ++ F+KACFSS R +S NL +S Sbjct: 182 PLKEKALASLAQAPFFEEALQKGFEMLEDFLKACFSSPRKTLSNNLKKS 230

____________________________________________________________________

(End of Fig. 6.2)

140

Of the five plasmids sequenced, four gave significant correlation to sequences on the

database. Plasmid pRU496 gave no correlation however the quality of the sequence

data was low.

Plasmid pRU529 had homology to a phytochrome from Synechocystis sp, having

34% homology over 70 residues followed further along by 34% over 41 residues.

Plasmid pRU499 showed significant homology to fixND from R. leguminosarum,

with 88% identity over 69 residues. Further study of the BLAST results indicated

that the homology is to fixN.

Plasmid pRU498 showed homology to metX (homoserine O-acetyltransferase) with

43% homology over 67 residues. This was one of two isolates that showed

significant increase in expression at day 10 compared with day 4. The other plasmid,

pRU530 was not sequenced. These constructs appear to be important in extreme

starvation.

Plasmid pRU500 has homology to a 16S rRNA dimethyltransferase with 40%

identity over 49 residues from Helicobacter pylori.

6.3 Discussion

Analysis of the library LB-6 was carried out to determine the effectiveness of the

libraries in general as well as the interesting constructs it potentially contains. As

such, the clones have only been partially characterised.

141

The library was initially selected for expression on agar plates with no expression

when grown in broth. In this chapter, further screening was carried out to identify

those strains with significant increase in fluorescence when grown on plates as

opposed to broth. Two constructs, pRU530 and pRU498, were identified with

increased expression late in stationary phase. It is proposed that these are important

to survival during extreme starvation. Plasmid pRU498 was sequenced and showed

homology to homoserine O-acetyltransferase (metX).

Plasmid pRU499 was homologous to fixN from R. Leguminosarum. This is a

component of CBB3 cytochrome oxidase complex and is expressed by bacteroids in

response to low oxygen. FnrN, a redox sensitive transcriptional activator,

homologous to E. coli Fnr, induces expression of fixN (Gutierrez et al., 1997). The

fixN gene is part of the fixNOQP operon found on pSym. Expression of FixN has

been shown to occur both in the symbiotic zone of the root nodule and under free

living microaerobic conditions (Schluter et al., 1997). When strain RU1183

(pRU499) was observed on plates, it was noticeable that expression only occurred in

single colonies and not on the main streak line where the colonies are amalgamated.

It could therefore be postulated that the microaerobic conditions necessary for

induction were not present in the streak line. Such expression is consistent with the

low oxygen tension found in mature single colonies (Lorian, 1989).

The homologies found for pRU529 and pRU498 were a 16SrRNA transferase and a

phytochrome respectively. The expression of these genes during growth on agar

rather than in broth is a mater for speculation. As the main function of screening this

library was to confirm the library construction, further study of these isolates has not

142

been carried out. They do pose some interesting questions and would be worthy of

future investigation.

One of the aims of the analysis of LB-6 was to confirm the successful construction of

the sub-libraries. Although this library has a small number of individual clones,

there is a wide range of expression conditions for the isolates. The sequence data

confirms that the inserts contain different DNA sequences with significant

homologies to known DNA. Some of the sequence homologies are also in keeping

with genes known to be expressed under these conditions. On the basis of this,

experiments were set up to identify environmentally induced promoters using IVET-

OT. This is described in Chapter 7.

143

Chapter 7

Isolation of Environmentally Induced Promoters using IVET-OT

7.1 Introduction

Previously the construction and testing of the optical trap has been described (see

chapter 4). A promoter library based on pOT1 and a sub library LB-3, containing

only the promoters that are below a low threshold on minimal laboratory media, has

been constructed (see chapter 5). In this chapter we carry out IVET-OT on the

library to identify promoters of Rhizobium that are induced by growth in the plant

rhizosphere. The complexity of the rhizosphere has prevented it being reproduced in

laboratory medium. Many factors, both biotic and abiotic, influence organisms in the

rhizosphere. In these trials, the environment has been simplified by growing pea

plants in vermiculite inoculated with a single strain. As the genetics of nodulation

are well characterised and these genes give an internal control in these experiments.

It would be expected that nod gene promoters could be recovered using this

technique. There are aspects of the Rhizobium-legume association that are poorly

understood. After rhizobia enter the root hair they are contained by the plant in an

infection thread, laid down between cortical cells. The genes expressed by rhizobia

during this period of nodule development are very poorly understood. IVET-OT

may enable genes specifically expressed in the infection thread to be examined,

however, the primary aim was to isolate genes expressed during colonisation of the

rhizosphere.

144

7.2 Isolation of LB-3 Clones from the Rhizosphere

Pea seeds, grown in universal bottles in N-free rooting solution (approximately

12ml), were inoculated with library LB-3. The inoculum for each plant was

calculated to theoretically contain 5 copies (approximately 60,000 cells) of the

complete library using the value 1.7x109cfu ml-1 (calculated from a dilution series

plating of LB-3) and was used directly from diluted thawed stock with no prior

growth. The plants were grown for 7 days before harvesting. Individual roots and

vermiculite were ground in N-free rooting solution (20ml) using a sterile pestle and

mortar. The cell suspension was filtered through a Whattman filter paper to remove

large particles, and 8ml centrifuged (8,500rpm, 30 sec). The supernatant was

centrifuged (13,000, 5 min) and resuspended (50µl N-free rooting solution, 7.5%

ficol).

The filtrate from the ground roots and vermiculite was observed under UV excitation

with the optical trapping microscope and 12 fluorescent cells were trapped, from four

separate plants, and isolated. The trapping media used was N-free rooting solution

with 7.5% Ficoll. The isolated cells were grown in TY broth (10ml supplemented

with gentamycin (20µg.ml-1) and streptomycin (500µg.ml-1). The conditions and

notes for the trapped cells are given in Table 7.1.

145

Cel

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nce

Plan

t

Lase

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rren

t

Atte

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ion

filte

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App

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tra

ppin

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N

o. C

ells

in

trap

Not

es

RI 1 1 13.5A None 30mW 2 Long time in trap

RI 2 1 13.5A None 30mW 1 High level fluorescence


RI 4 2 13.5A None 30mW 2 Long cell - Dividing?

RI 5 2 13.5A None 30mW 1 High level fluorescence visible after trapping

RI 6 3* 13.5A None 30mW 1 Low level fluorescence


RI 8 3* 13.5A None 30mW 1 Low level fluorescence - Only moved 2mm


RI 10 4 13.5A None 30mW 1 Still fluorescent after trapping


RI 12 4 13.5A None 30mW 2 Large dividing cell

It was noted that plant 3 had no high level fluorescent cells in its extract. The cells

trapped were fluorescent but the levels were not as high as the cells isolated from the

other plants. This could be due to variation in the plant growth however even simple

contact with the root system would be expected to induce some genetic response.

Table 7.1 Summary of the cells trapped after exposure to plant roots. If more than one cell was trapped this is noted. * The cells within the extract from plant 3 were not highly fluorescent.

146

7.3 Characterisation of the Rhizosphere Isolates.

Twelve cells were trapped, of which four grew in TY broth (Gentamycin 20µg.ml-1,

streptomycin 500µg.ml-1), RI 3, RI 5, RI 8, and RI 10 (Table 7.2). To establish if

these cultures had grown from a single trapped cell or were contaminated with other

cells, a PCR screen was devised. The cultures were plated out and 6 individual

colonies from each were PCR amplified using the colony boiling method with

primers P80 and P12 (See Fig. 5.2). If the cultures grew from several cells, more

than one PCR profile would be expected. A set of 6 identical PCR fragments

indicates that contamination of the culture is less than 1:6 colonies. Due to the

isolation procedure, a culture contaminated by other library constructs would be

expected to contain few cells originating from the isolated cell and the majority of

clones would be from other library constructs. It can therefore be assumed that if all

6 PCR profiles are identical, they probably originate from the trapped and isolated

cell. Of the four cultures that grew, three had 6 identical PCR profiles (Table 7.2).

Isolate RI 5 gave 6 different PCR fragments from 6 colonies. This culture also grew

in less than 3 days, whilst the others took approximately 5 days, which also indicated

that the culture, RI 5, originated from more than one cell.

Construct No. of days for initial growth of culture

Potentially clonal Insert size

RI 3 7 days Yes 3.2 Kb

RI 5 < 3 days No N/A

RI 8 7 days Yes ~0.4 Kb

RI 10 5 days Yes ~0.4 Kb

Table 7.2. Summary of the constructs that grew after trapping. The Culture was considered to have originated from a single isolated cell if all 6 PCR reactions gave identical size bands. RI 5 gave 6 different sized PCR products.

147

The three strains derived from single cells were reinoculated onto pea plants and on

AMA (10mM glucose, 10mM ammonia). After 7 days extracts were made and

observed using fluorescence microscopy. RI 10 gave no fluorescence from either

plates or from the root extract. Almost all the cells from the root extract of RI 8 were

moderately fluorescent, however, when a colony from the plate was observed

microscopically, a similar level of fluorescence was observed. RI 3 gave high level

fluorescence from the root extract with only low level from the plates.

The strain RI-3 was originally isolated as being fluorescent in the presence of plant

roots from a population that was non-florescent on minimal laboratory media. From

this it appears that RI-3 has been successfully isolated with a promoter region that is

induced in the presence of a plant root. Once purified RI-3 was reinoculated onto

plants and became fluorescent at a high level in most of the observed cells when

exposed to plant roots but was non-fluorescent on minimal laboratory media. It thus

fulfils Koch's postulates. This isolate was designated as strain RU1302 and plasmid

pRU504 isolated from it using FlexiPrep kit (Pharmacia).

DNA sequencing was carried out from pRU504 using cy5 labelled pOT-reverse

primer. This primer is designed to sequence from the 5' end of gfpuv into the MCS

of pOT1, thus sequencing the inserted genomic DNA. BLAST searches (performed

at the GSC using the BLAST network service) on the sequence showed homologies

to thiamine phosphate pyrophosphorylate (thiE) from Archaeglobus fulgidus: 39%

identity over 92 residues (Bases 290 to 15) (Fig 7.1). The next sequence homology

148

was to Hydroxyethylthiazole (thiM) from E. coli: 33% homology over 99 residues

(bases 714 to 338). The only homology for this data with a Rhizobium strain was the

27th listed result. This was to R. meliloti (32% homology over 118 residues (Bases

110 to 451) and was not homologous to any known sequences.

To assist with further analysis the PacI fragment (3.2Kb) from pRU504 was cloned

into pNEB193 to give pRU569. DNA sequencing was carried out on this plasmid

and again showed homology to thiE when sequenced from the reverse primer. There

were no significant homologies when sequenced from universal primer indicating

that the 3.2Kb insert in pRU504 (pRU569) extends beyond the thi genes at the end

distal to gfp. From the Reverse primer there was a homology with thiE from R. etli.

However, the homology was low: 42% over only 40 residues. This was the 14th

listed result of 15 results (Fig. 7.1).

The restriction analysis and open reading frames for pRU504 are shown in Fig 7.2.

Open reading frame analysis of the sequence revealed three open reading frames.

One coincides with the thiM sequence however the program did not find an open

reading frame for thiE. The two genes however are not in the same reading frame

and due to the number of ambiguous bases, the program appears to have missed the

true end of thiM. Observing the sequence data an ATG at base pair 305 identified

the open reading frame for thiE in the -3 reading frame.

149

1 GATGTCGHXA TCGCCcGCGC TCTCATCGCA TCCATGTCTT CCTGGCCGAT 51 ATGCAGGCCG TCGACGCCGA TGGCGATTGC CGCCTCGACG TCATCGTTGA 101 CGATGAGCAG GGCGCCGGTC CCATCCAGCG CCTGTTTCAA GGCGCGGCCG 151 GTCTCGATCA TCCTGATGGT GCCGGCATGT TTGTCACGCA ACTGCACCAT 201 GGTCGCGCCG CCGGCAACGG CAAGGCGCGC GGTTTCGACC ATGCCGATCC 251 CGGCGCAGAG ATCGGGGTCG AGGACGAGAT AGAGCGAAAG GTCGAAAGCC 301 TTCATGCAGC TGATATCCTT GCCCTGGCGT CAAGCGTTTC GGCGTCGAGC 351 GCGGCCAGCG CATCGAGAAA ACGCCAGGAG AAGGAGCCGG GCCCGGCCGC 401 CCCAAGGGCT GCCTCCTCGC CGGCGATGGC GAAGGYTGMW MMGTGCAGCG 451 ACCcGTCGCG CCGAMGATAT CCTCTGGCGC CGTCGCGGCA AARGCGCCGA 501 CGAGGCAGGT GAGCGAGCAG CCGAGCGCGG TGACCTGAGG CATCAAGGCC 551 GATCCGCCTT CGATGCGCAC CGCCCGCTCG CCGTCGGTGA CGAAATCCAC 601 GGCACCGGTG ACGGCAACCA CCGCCCGCTG CCGCTTCAGC CAGACCATHG 651 CGCCGvAACC TTCCSCCTST TyCGAACCGG ATCGCSGGCT GTTSGACGCC 701 CTGGCCACGG GbTTTYTCCC SCCGGCAAGC GCGCDcATYT CcSGACGCGY 751 TBCCCXcGCD AWYSYt

Translation of Open reading frame -3 from the start site at base pair 305. This is

homologous to thiE (Bold above):

MLAFDLSLYL VLDPDLCAGI GMVETARLAV AGGATMVQLR DKHAGTIRMI ETGRALKQAL DGTGALLIVN DDVEAAIAIG VDGLHIGQED MDAMRAR

Fig 7.1 The sequence and BLAST results of the cloned promoter fragment in pRU504. The second BLAST results are for the same region cloned into pNEB193 (pRU569). For ambiguity codes see Fig. 6.2

150

BLAST results 1 and 2 for pRU504:

trembl|O28205 (AF2074) THIAMINE PHOSPHATE PYROPHOSPHORYLASE (THIE). [ARCHAEOGLOBUS FULGIDUS] Length = 210 Minus Strand HSPs: Score = 165 (58.1 bits), Expect = 9.4e-12, P = 9.4e-12 Identities = 36/92 (39%), Positives = 51/92 (55%), Frame = -3 Query: 290 LSLYLVLDPDLCAGIGMVETARLAVAGGATMVQLRDKHAGTIRMIETGRALKQALDGTGA 111 LS+Y + D + G E A +A+ G +Q R+K T RM E G+ L+ A Sbjct: 8 LSVYFITDSEF--GRTHEELAEMALRAGVRAIQFREKKLSTKRMYEIGKRLRALTRDYDA 65 Query: 110 LLIVNDDVEAAIAIGVDGLHIGQEDMDAMRAR 15 L VND ++ A+A+ DG+HIGQ+DM A AR Sbjct: 66 LFFVNDRIDVALAVDADGVHIGQDDMPAFAAR 97

sp|P76423|THIM_ECOLI (THIM) HYDROXYETHYLTHIAZOLE KINASE (EC 2.7.1.50) (4-METHYL-5-BETA- HYDROXYETHYLTHIAZOLE KINASE) (THZ KINASE) [ESCHERICHIA COLI] Length = 262 Minus Strand HSPs: Score = 133 (46.8 bits), Expect = 8.5e-10, Sum P(2) = 8.5e-10 Identities = 33/99 (33%), Positives = 47/99 (47%), Frame = -2 Query: 741 EMXALAGGXXPVARASNSXRSGSXXXEGXGAMVWLKRQR-AVVAVTGAVDFVTDGERAVR 565 E+ ALAG +A + A L R+ A+V VTG +D+VTDG R + Sbjct: 130 EIMALAG----IANGGRGVDTTDAAANAIPAAQTLARETGAIVVVTGEMDYVTDGHRIIG 185 Query: 564 IEGGSALMPQVTALGCSLTCLVGAFAATAPEDIXGATGRC 445 I GG LM +V GC+L+ +V A A + + C Sbjct: 186 IHGGDPLMTKVVGTGCALSAVVAACCALPGDTLENVASAC 225 Score = 66 (23.2 bits), Expect = 8.5e-10, Sum P(2) = 8.5e-10 Identities = 16/29 (55%), Positives = 17/29 (58%), Frame = -1 Query: 424 AGEEAALGAAGPGSFSWRFLDALAALDAE 338 AGE A + GPGSF FLDAL L E Sbjct: 231 AGERAVARSEGPGSFVPHFLDALWQLTQE 259

------------------------------------------------------------------------------------------------------ 27th result for pRU504:

trembl|Q52921 NOT HOMOLOGOUS TO KNOWN SEQUENCES AS OF 2/92. [RHIZOBIUM MELILOTI] Length = 208 Plus Strand HSPs: Score = 98 (34.5 bits), Expect = 0.028, P = 0.027 Identities = 38/118 (32%), Positives = 49/118 (41%), Frame = +2

151

Query: 110 GRRSHPAPVSRRGRSRSS*WCRHVCHATAPWSRRRQRQGARFRPCRSRRRDRGRGRDRAK 289 GR + P P+ R RS ++ W SRR +GAR R RRDRGRG + + Sbjct: 23 GRCAAPRPLVRSARSHAAAWRPRDRGDGGRNSRRADAEGARHH--RRLRRDRGRGDPQGR 80 Query: 290 ---GRKPSCS*YPC-PGVKRFGVERGQRIEKTPGEGAGPGRPKGCLLAG---DGEGCXVQ 448 G +P PC + +R R + PG R GC G GC Q Sbjct: 81 QPGGARPLPD-RPCFKRARSHAGDRETRQDTAPGSRHAAWRRHGCGSCSRHRQGGGCLPQ 139 Query: 449 R 451 R Sbjct: 140 R 140

____________________________________________________________________

BLAST result 1 for pRU569:

tr|O28205 (AF2074) THIAMINE PHOSPHATE PYROPHOSPHORYLASE (THIE). [ARCHAEOGLOBUS FULGIDUS] Length = 210 Score = 47.6 bits (111), Expect = 4e-05 Identities = 22/49 (44%), Positives = 30/49 (60%) Query: 202 MIETGRALKQALDGTGALLIVNDDVEAAIAIGVDGLHIGQEDMDAMRAR 56 M E G+ L+ AL VND ++ A+A+ DG+HIGQ+DM A AR Sbjct: 49 MYEIGKRLRALTRDYDALFFVNDRIDVALAVDADGVHIGQDDMPAFAAR 97

------------------------------------------------------------------------------------------------------

14th result for pRU596:

tr|O34294 (THIE) THIAMIN PHOSPHATE PYROPHOSPHORYLASE (EC 2.5.1.3) (THIAMIN-PHOSPHATE PYROPHOSPHORYLASE) (TMP PYROPHOSPHORYLASE). [RHIZOBIUM ETLI] Length = 204 Score = 31.7 bits (70), Expect = 2.7 Identities = 17/40 (42%), Positives = 21/40 (52%) Query: 187 RALKQALDGTGALLIVNDDVEAAIAIGVDGLHIGQEDMDA 68 R K A LI+ND AI G D +H+GQED+ A Sbjct: 44 RRAKAACAAAACQLIINDYWRLAIDEGCDFIHLGQEDLMA 83

____________________________________________________________________

(End of Fig 7.1)

152

A) Computer generated restriction map of pRU504 insert (thiE thiM genes) 766bp

Aat II (92) Acc I (62)

Age I (605)

Alu I (310)

Alw NI (132)

Aos I (567)

Apa I (394)

Asp 700 (299)

Ava II (118) Avi II (567)

Ban II (394)

Bbs I (30)

Bcg I (184)

Bmy I (394)

Bpu AI (30)

Bsa I (157)

Bsm AI (157)

Bsm FI (104)

Bsp 120I (390)

Bsp 1286I (394)

Bsp MI (497)

Bsr BI (577)

Bst EII (530) Bsu 36I (536) Dde I (536)

Eco 57I (620)

Fsp I (567)

Mae II (89)

Mbo II (30) Mlu NI (705)

Msc I (705)

Msl I (164)

Nco I (198)

Nsp I (180)

Pin AI (605)

Pvu II (310)

Sal I (61)

Xmn I (299)

B) Open reading frame analysis of pRU504 insert (thiE thiM genes) 766 bp. Thin lines show computer analysed open reading frames. Bold line shows thiE open reading frame from sequence data analysis.

Nco I (198)

thiM

thiE 305

Fig. 7.2 Restriction map (A) and open reading frames (B) in the insert in pRU504

153

The gene thiE is implicated in the final stages of thiamine biosynthesis (Fig 7.3).

This implies that this gene was switched on due to thiamine deficiency in the media

surrounding the root. To confirm this, RU1302 should be plated on media deficient

in thiamine. This however proved problematic, as Rhizobium grows very poorly

without this vitamin. Due to time constraints this has therefore not yet been

confirmed.

PRPP PRA AIR AICAR

thiO? thiG thiCc

THZ-P thiE HMP-P

IMP

THIAMIN GMP AMP

7.4 Discussion

The library LB-3 was selected to contain pOT1 clones with R. leguminosarum

random genomic DNA fragments whose expression was off during growth in

minimal broth. IVET-OT was carried out on this library by exposing it to the

rhizosphere of pea plants. Twelve cells, expressing GFP, were trapped and four of

these were cultured. One strain, RU1302 (pRU504) was identified as expressing

GFP only when exposed to the rhizosphere. The cloned DNA was sequenced and is

homologous to thiE from a number of bacterial strains with homology to E. coli thiM

further along the sequence.

Fig. 7.3 The purine and thiamine biosynthetic pathways in R. etli [Miranda-Rios, 1997 #122] AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide THZ-P: phosphated thiazole HMP-P: phosphated hydroxymethylpyrimidine AIR: 5-aminoimidazole-ribonucleotide

154

The gene thiE is implicated in thiamine biosynthesis, specifically in the condensation

of phosphated hydroxymethylpyrimidine and thiazole to synthesise thiamine

phosphate (Backstorm et al., 1995). Thiamine is one of three vitamins added to

minimal growth media for Rhizobium (AMS and AMA). Pantothenate can be

removed from the growth media and R. leguminosarum still grows, however, if

biotin or thiamine is removed, R. leguminosarum grows poorly. Four genes, thiC,

thiO, thiG and thiE, have been sequenced and analysed from R. etli (Miranda-Rios et

al., 1997). These are found on the plasmid pb and sequences submitted to GenBank

(accession no. AF00448). It is notable that the sequence from pRU504 shows low

homology to that of thiE from R. etli strain CFN037 (Miranda-Rios, et al., 1997).

This mutant strain has a Tn5mob insertion in the thi box preceding thiCOGE. The

thi box is a 39bp conserved region found upstream of a number of thi genes. It has a

proposed function in regulation of these genes as the mutation in CFN037 has

constitutive expression of at least thiC, whilst the wild type does not. This thiCOGE

gene locus is located on plasmid pb. A strain with the pb plasmid cured, CFNX183,

is unable to grow on minimal media without thiamine, however thiamine

biosynthesis is complemented by the thiCOGE locus. This demonstrates that for R.

etli no other genes necessary for thiamine biosynthesis are found on the pb plasmid.

Strain 3841, from which the insert in pRU504 was derived, is unable to grow on

minimal media without addition of thiamine. It is notable that the sequence from

pRU504 only has a low homology to the CFNX183 thiE. This suggests that the thiE

in our study may be a chromosomal thiE gene. The R. etli strain may grow better on

thiamine deficient media if it has a chromosomal as well as the plasmid copies of the

thiamine biosynthetic genes. Rhizobium 3841 may not contain the plasmid copy,

155

resulting in poor growth. It may be that some rhizobia have two copies of the thiE

gene to increase thiamine biosynthesis, however this is speculative and requires the

identification of two thiE genes from one organism.

Thiamine biosynthesis may have an additional role in the symbiosis of Rhizobium.

From the analysis of R. etli mutations of the purine biosynthetic pathway, it has been

suggested that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) is a

negative effector of symbiotic terminal oxidase (Soberon et al., 1997). It has also

been shown that AICAR or a related metabolite promotes the development of the

infection thread (Newman et al., 1995). In turn this pathway is metabolically linked

to thiamine biosynthesis (Fig. 7.2). The mutant CFN037 was originally isolated by

its increased capacity to produce cytochrome c terminal oxidases. The genes

encoding terminal oxidase in several Rhizobium species are fixNOQP, which are

expressed in response to low oxygen. As ThiC is involved in the synthesis of 4-

amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) from the purine

intermediate 5-aminoimidazole-ribonucleotide (AIR), and this is also a precursor of

AICAR, increased expression of thiC in turn effects AICAR (Fig. 7.3). In turn,

AICAR or a related metabolite is acting as a negative effector of fixNOQP

expression. Mutant CFN037 is able to fix more nitrogen than the wild type during

symbiosis.

This suggests an important role for vitamins in symbiosis and raises several

questions. Rhizobium leguminosarum normally lives in the soil, a vitamin deficient

environment, however it is unable to grow on laboratory media without additional

thiamine and biotin. It could be postulated that Rhizobium is in some way tied into

156

the rhizosphere by the need for vitamins. The plant could be providing these

vitamins or some other factor that affects their biosynthesis. Biotin uptake and

synthesis has been shown to contribute to successful colonisation of R. meliloti

(Streit, et al., 1996). Plant derived biotin promotes the root colonisation, however

biotin synthesis is the more important source. Biotin supplied by the plant could be

promoting the biosynthesis or uptake of biotin within the cell, transforming

Rhizobium from a slow growing to a fast growing state. A biotin regulated locus,

bioS, has been identified and could be implicated in this system (Streit and Phillips,

1997). Thiamine is also an essential vitamin, limiting Rhizobium growth and

released by plants, and a similar regulatory system could be envisaged.

Further work is needed to establish the relevance of thiE and thiM in pRU504. The

genes need to be mutated in R. leguminosarum 3841 to determine their effect on the

cell. The expression of these genes should be determined in the absence of thiamine.

As this is not possible during growth, RU1302 could be grown on minimal media

containing thiamine and resuspended in thiamine deficient media. The expression of

the gfpuv fusion could then be monitored. It would also be important to establish the

available thiamine in the soil and rhizosphere and the effect this has on expression in

RU1302.

In this chapter we have demonstrated the IVET-OT system and shown that

environmentally induced promoters can be identified. This strategy is powerful in its

ability to identify environmentally induced promoter. However, it shares a limitation

with other IVET protocols, in identifying what promoter level is necessary to get

expression. In the original IVET strategy, a certain level of expression is needed to

157

rescue growth. In IVET-OT, the ability to observe fluorescence is restricted to a

certain expression level. In this case GFPuv is superior to wtGFP as it is 35x more

fluorescent. This could be further studied using the control strains in isolated during

library construction (Chapter 5). Another problem in IVET-OT is identifying

transiently expressed genes. IVET is unable to identify these genes. IVET_OT picks

up these genes as GFPuv is stable, thus once expressed the fluorescent remains even

after the gene promoter activity has ceased. The problem is in rescreening

transiently expressed promoters. By definition the promoter is only on under specific

conditions and so inoculating pea plants with a purified clone will result in only a

few fluorescent cells. This may be the case for isolate RI 10, where bacteria from

root extract inoculated with purified RI 10, were not fluorescent. The ability to

obtain spatial information about expression and to trap bacteria directly from the

environment may be crucial in obtaining transiently expressing isolates. Peas could

be inoculated with purified isolates and the root system observed under the

microscope. If expression is limited to a particular location such as the infection

thread, the cell could be re-trapped, confirming the promoter involved.

158

Chapter8

Discussion

An understanding of the genetic processes is essential in the study of the interaction

between a symbiant and its host. The environmental factors effecting Rhizobium as

it develops from a free-living soil organism, to residing in the rhizosphere and its

eventual nodulation of legume roots is complex. Many of the processes are not well

understood at a genetic level. In an attempt to identify these environmentally

induced genes an adapted IVET strategy was proposed utilising optical trapping

(IVET-OT). An important aspect of this strategy is to be able to identify promoter

activity in the environment and isolate individual cells exhibiting that activity

without the need to grow the cell on laboratory media prior to isolation. In this way

promoters induced by complex environmental stimuli can be isolated. To this end an

optical trap was designed, built and developed along with a plasmid promoter probe

vector in which genomic libraries can be expressed.

The optical trap was based on an Optiphot epifluorescence microscope (Nikon) and a

Nd Yag laser (1064nm). The microscope was modified to incorporate the laser.

This had a detrimental effect on the optics, reducing image quality slightly and

removing the ability to change objective lenses without refocusing. This however

was not a significant problem as the microscope was dedicated to optical trapping.

The laser output power had to be attenuated to power levels survivable by the

bacteria (Approximately 30mW). This was achieved by reflecting the majority of the

laser to a heat-sink using an 85% reflective mirror. Since the construction of this

trap, low power lasers, operating at 1064nm, have become available on the market

159

that could be used for this work. These have the advantage that they are small

making them easier install and work at milliwatt output levels removing the need for

attenuation. They also work from the standard 13Amp power rather than expensive

3 phase power source and use air cooling rather than water-cooling. This makes

them cheaper to run and more portable.

The optical trap is a powerful tool allowing the isolation and study of single cells.

When used in IVET-OT the spatial information given by using the microscope, in

combination with the ability to isolate a specific cell, makes this a unique tool in

identifying gene expression. Once gene libraries are constructed, the simplicity of

using the optical trap makes the identification of large numbers of specific gene

fusions relatively simple. The limiting step in this technique is the characterisation

of the isolates. The power and adaptability of optical trapping as a tool is likely to

make it a common piece of equipment in many laboratories.

Using this trap we demonstrated that about one in three cells trapped could be

cultured. The cells that were not cultured could represent dead cells. Alternatively

they may be damaged during trapping or some other factor or signal may be

preventing growth. Further study is needed on the effects of optical trapping on

individual cells. It is known that trapping effects lipid membrane causing vesicles to

burst (Bar-Ziv, et al., 1995). The trap may also be effecting ion exchange or

transport across the membrane.

A series of promoter probe vectors were designed and constructed culminating in the

construction of pOT1. This has a broad host range replicon (pBBR1-MCS-5

160

(Kovach, et al., 1995)) and a gentamycin resistance marker. The reporter fusion is

transcriptionaly isolated from the replicon by two transcriptional terminators with

different sequences. The reporter, gfpuv, has a bacterial ribosome binding site and a

multiple cloning site (MCS) for the insertion of genomic DNA. The vector has been

designed in a modular fashion making it possible to adapt the plasmid. It would be

possible to use this vector in FACS sorting of genomic libraries using a red shifted

GFP (Cormack, et al., 1996; Crameri, et al., 1995). However the use of the optical

trap allows specific individual cells to be isolated from a complex environment.

A library (LB-2) was constructed by cloning R. leguminosarum genomic DNA into

pOT1. Due to time constraints we achieved approximately 40% coverage of the

genome. It would be valuable to construct a complete genome library in this vector.

Two important sub-libraries were defiled from LB-2. The first, LB-3, consists of

clones that are off when grown in minimal broth and agar. The second, LB-6,

consists of clones that are off in minimal broth but have increased fluorescence on

minimal plates. Further sub-libraries, LB-4 and LB-5, were also isolated. These

consisted of clones on the borderline of being members of LB-2 and were separated

to ensure that all members of LB-2 were off under minimal growth conditions. LB-4

and LB-5 can be used in a similar way to LB-3.

Comparison of the fluorescence of LB-6 constructs on minimal plates and growth

lead to the isolation of 24 clones whose expression was at least three times higher on

plates than in broth. This included two strains, RU1256 and RU1188 whose

expression increased during late stationary phase (10 day old culture). A number of

the isolates were analysed. RU1216 (pRU529) has homology to phytochrome;

161

RU1188 (pRU498) has homology to homoserine O-acetyltransferase (metX);

RU1184 (pRU500) has homology to 16S rRNA dimethyltransferase; RU1183

(pRU499) has homology to a component of a terminal cytochrome oxidase (fixND).

After exposure to pea roots, LB-3 was recovered and fluorescent cells isolated by

optical trapping. In this first demonstration of IVET-OT four cells were successfully

cultured from 12 trapped cells. One of these, RU1302, was induced only in the

presence of roots. When the purified strain was reinoculated onto pea roots, almost

all recovered cells were highly fluorescent. One of the problems associated with

IVET-OT is the characterisation of transiently expressed genes. Unlike the original

IVET system, transiently expressed genes are recovered in the population obtained

from the roots. Once GFPuv matures, fluorescence is stable and maintained for long

periods of time. Transient expression at any time during exposure to the

environment can be identified within the sensitivity of GFPuv. However, once

purified, a transiently expressing strain is harder to characterise. The sensitivity of

GFPuv, although high, still needs to be quantified with respect to promoter activity

for this system. Characterisation of the fluorescent standards identified in chapter 5

(Table5.1) could be used to evaluate this. By definition a transiently expressing gene

may only be expressed at a certain point in the lifecycle and for only a specific time.

Reinoculating a plant with the strain will not result in large numbers of fluorescent

cells. To overcome this it may be necessary to observe the root system over an

extended period of time during growth and development. This could be achieved by

growing roots between microscope slides, allowing periodic observations.

Alternatively roots and nodules could be sectioned to identify bacteria expressing

GFPuv specifically within these tissues. Another method for identifying transiently

162

expressed promoters would be to use FAC scanning. If a purified isolate from IVET-

OT appeared non-fluorescent when reinoculated onto pea roots, the retrieved cells

could be compared to a control using FAC screening of a large number of cells

(10,000 plus) in a very high fluorescence window. An increase of cells falling in that

window from, for example 0.01% to 0.1% would indicate a significant increase in

fluorescent cells. This change would not be observable using a microscope but

would indicate transient expression of the promoter in that isolate. These techniques

warrant further investigation.

The genomic insert in plasmid pRU504 (isolated from RU1302) was homologous to

thiE and thiM. These genes are important in biotin biosynthesis and, by a metabolic

link through AICAR, to symbiosis (Chapter 7). As R. leguminosarum is unable to

grow on minimal media without thiamine, it would appear that sufficient induction of

thiamine biosynthesis is not triggered by the simple lack of thiamine. This implies

that another factor is missing or that this strain is a thiE auxotroph. The induction of

thiE in the rhizosphere may be important in stimulating growth in the rhizosphere

prior to infection of the root. A mechanism could be proposed where Rhizobium is

maintained in the soil with little or no growth by the lack of vitamins. Once in the

rhizosphere a factor, possibly plant derived, induces the thiamine biosynthetic

pathway allowing growth and eventual root infection. Alternatively the plant may

supply thiamine in sufficient quantity for growth. To investigate the function of thiE

in R. leguminosarum, the genomic locus needs to be identified and mutated. The

mutant could be tested for its ability to grow on thiamine deficient media, and its

ability to nodulate and fix nitrogen during symbiosis. Similarly constitutive

expression of the gene could be investigated.

163

In this project, the IVET-OT technique has been developed and environmentally

induced genes have been isolated. The system is powerful in it ability to identify

these genes and it is anticipated that other important genes will be identified using

this method.

164

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development of optical trapping for the isolation of ......vetch roots. other rhizobium strains...

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