Technical University Braunschweig

German Research Centre forBiotechnology, Braunschweig

WP5

Oil - degradative capacities

Sample chosen: U3

Objectives

Evaluation of major microbial groups

Background for applications

Adjusting protocols

2. 16S rRNA gene libraries

Oil issue:

1. Enrichment with crude oil, isolation

3. Metagenomes:“Archive” libraries, Expression libraries

Three pathways for the metabolic attack on n-alkanes are known:

Molecular genetics and pathway for alkane oxidation

ii) biterminal oxidation ( of several types of bacteria and fungi)

H3CRCH3 ->H3CRCH2OH -> HOCH2RCH2OH -> HOOCRCOOH

iii) subterminal oxidation (Nocardia spp.) RCH2CH3 -> RCH(OH)CH3 -> RC(O)CH3

i) monoterminal oxidation pathway (Pseudomonas spp.)

RCH3 -> RCH2OH -> RCHO -> RCOOH

Molecular genetics and pathway for alkane oxidation

Enrichment (Urania interphase)

Pure cultures

Dilutions, plating

Metagenomes

DNA prep

PCR, cloning

Re-amplification

RFLP-Clustering,sequencing sequence analysis

Oil enrichment "O": selected for sequencing1 2 3 4 5 6 7 8 9 10 11 12

A 1 2 3 4 5 6 7 8 9 10 11 12B 13 14 15 16 17 18 19 20 21 22 23 24C 25 26 27 28 29 30 31 32 33 34 35 36D 37 38 39 40 41 42 43 44 45 46 47 48E 49 50 51 52 53 54 55 56 57 58 59 60F 61 62 63 64 65 66 67 68 69 70 71 72G 73 74 75 76 77 78 79 80 81 82 83 84H 85 86 87 88 89 90 91 92 93 94 95 96

Incubation for six weeks at r.t.

0.1 substitution/site

Thiomicrospiragroup

Chromatiaceae

Legionellaceae

CardiobacteriaceaeMethylococcaceaeEnterobacteriaceaePasteurellaceae

Vibrionaceae

Aeromonasgroup

Colwelliagroup

Xanthomonas- Stenotrophomonas group

Microbulbifer

Neptunomonas

Marinomonas

Alcanivorax

Oleiphilus messinensis

Oceanospirillum

Marinobacter

Pseudomonas(sensu stricto)

Isolates from oil enrichment.

Ps. stutzeri-Ps. balearica groupMarinobacter hydrocarbonoclasticus-Mb.CABProteobacteria (Ruegeria group)

METAGENOMIC LIBRARIES

pBAC (e.g. pBeloBac11) large fragments 50-300 kbp

SuperCos1 - a „common“cosmid for cloning, Fragments to clone 40-50 kbp

“Archives”

based systems, e.g. LambdaZap

Expression libraries

pLAFR3 - a replicon in a variety of Gram-negatives(e.g. P. putida)fragments to clone - 20-30 kbp

SuperCos1 cosmid

Strategy to construct genomic library in cosmid vectors pLAFR3/SuperCos

Cosmid arms treated with phosphatese

Transduce, select for antibiotics resistans and score for white colonies on X-Gal

DNA size-fractionated,partially digested with Sau3A

Ligation

Package in vitro

Library of dozens of thousands arrayed clones with 20-50 kbp inserts

Cosmid library (contd.)

Arrayed clonesColony picking

Sequencing of selected clones

Subcloning/expres-sion of gene of interest

Probing

Enzymes Detection systemEsterases/proteases

Phosphatases

Sulfatases

Esterases/proteases

Lipases

Phosphatases

-D-Galactosidases

-D- Galactosidases

-D-glucosidases

Indoxyl acetate

Indoxyl phosphate

Indoxyl sulfate

naphtyl acetate/butyrate+FastBlueRR

naphtyl palmitate + ””

naphtyl phosphate + ””

naphtyl -D-galactopyranoside + ””

naphtyl -D- galactopyranoside + ””

naphtyl -D-glucopyranoside + ””

Enzymes and detection systems

The ZAP Express vector allows bouth eukaryoticand prokaryotic expression and accomodates DNA insert from 0 to 12 kb in length.

„Oil“ library = 1,8 x 106 phage particles. Average insert size - 7.5 kbp

Clones in the ZAP Express vector can be screend with either DNAprobes or antibody probes

Phage expression system

Insert cloned into the ZAP Express vector excised out of the phage in the form of the Km-resistant pBK-CMV phagemid vector

Screening of ca. 10000 phage clones yields ca. 20 positives

Excision

Selected clones clustered

500 600 700 800 900 1000 1100

20

40

60

80

100 518.3

569.2598.1

637.2

673.4711.3

775.3

826.0

867.0925.5

994.71035.4

Expression

MALDI-TOF

Purification

Sequencing of selected clones

Product, enzymology

From phage library to enzyme

A short DNA probe anneals to a target DNA of interest. The probe then acts as a primer for a Rolling Circle Amplification reaction

1

The free end of the probe anneals to a small circular DNA template. A DNA polymerase (white oval) is added to extend the primer.

2

The DNA polymerase extends the primer continuously around the circular DNA template generating a long DNA product that consists of many repeated copies of the circle.

34

By the end of the reaction, the polymerase gene-rates many thousands of copies of the circular template, with the chain of copies tethered to the original target DNA. This allows for spatial reso-lution of target and rapid amplification of the signal.

RCA

Polymerase of phage

Perfectly works with small circular templates

Requires improvement of reaction conditions for whole-genomeamplification

Oil enrichment of a selected sample yielded “typical” marine HC-degraders, Marinobacter spp. and pseudomonads

Isolation will be continued with other samples/enrichmentsThose enrichments will be analysed for taxonomy of populations

Conclusions and outlook

The techniques used make possible to detect the enzymatic activities in metagenomes (dozens of clones per single activity screening)

Isolation of a variety of enzymes will be continued

Lack of material. To make a compehensive library, few dozens of micrograms of DNA or some dozens miligrams of the fresh biomass are required. Large-scale samplings of max. 1-2 sites (min 200 L) are needed