CHAPTER

ONE

1.1 INTRODUCTION:

Changes in environmental growth conditions may often be detrimental to survival and growth,

and in the course of biological evolution, all organisms have acquired a battery of mechanisms to adapt

to such changes in their growth conditions. The unicellular bacterium, Escherichia coli, too possesses a

variety of defense mechanisms to tolerate and to be able to grow under various stress conditions, of

which osmotic stress is one.

1.2 OSMOREGULATION:

All living cells share the common property of being bounded by a selectively permeable mem­

brane which acts as a barrier to most solutes, and is readily permeable to water. Hence changes in the

external solute concentrations have the same physicochemical (ie., osmotic) effects on all cells. A change

in external osmolarity causes net movement of water from the region of lower solute concentration (more

precisely, higher water activity) to that of higher solute concentration and as a consequence, there are

changes in cell volume and in intracellular solute concentration which could affect growth of the organ­

isms. Osmoregulation refers to the active processes carried out by cells in order to adapt to and grow in

an environment of changing water activity.

The higher animals (including man) possess highly evolved mechanisms to adapt to changes in

water activity of their environment. The osmolarity of the "internal milieu" (ie., the extracellular fluid bath­

ing all tissues) is maintained more or less constant and isotonic with respect to that inside the cells; these

cells lack a rigid cell wall. On the other hand, plants and bacteria are both bound by a rigid cell wall that is

situated external to the semipermeable cytoplasmic membrane. The latter two also differ from cells of

higher animals with respect to osmoregulation, in that each cell is directly affected by changes in osmolar­

ity of the environment. There exist remarkable similarities between both these groups of organisms in

their cellular responses to hyper-osmotic stress, in that they both accumulate the same set of organic and

inorganic molecules, known as compatible solutes.

1

The bacterium £. coli has a cell membrane and a cell wall surrounding it. The cell wall, but not

the cell membrane, is permeable to most solutes, and it is thus the cell membrane that acts as a barrier

which partitions the cell interior from cell exterior. £. coli cells maintain an intracellular osmolarity higher

than that of their surroundings, thereby generating a mechanical (hydrostatic or osmotic) pressure against

the cell membrane, called turgor pressure (21). Turgor pressure is an outwardly directed pressure and its

maintenance has been proposed to provide the mechanical force for expansion of the cell wall during

growth (122).

Maintenance of intracellular volume and of turgor pressure is extremely important for various

metabolic functions and growth. In an environment of fluctuating osmolarity, £. coli cells maintain a

constant volume and turgor pressure [of about 300 kilo Pascals (11 0): 1 atmosphere or Bar= 1 o5 Pas­

cals; and 22.4 atmosphere = 1000 mosmolar (mOsm) at NTP] by active processes that are collectively

known as osmoregulation.

In eukaryotes, the main experimental approaches for studying osmoregulation have necessarily

been physiological ones. Prokaryotic osmoregulation, however, is amenable to genetic analysis, which

therefore make bacteria excellent model systems for studying osmoregulation. £. coli (as well as the

closely related bacterium Salmonella typhimurium) has been used extensively over the decades in genet­

ic studies on many fundamental biological processes. When taken together with the fact that this organ­

ism is able to survive and grow in a wide range of osmolarities (varying from 0 to about 1200 mOsm), the

utility of £. coli as a model system to investigate the mechanisms of osmoregulation becomes apparent.

1.3 EFFECT OF HYPER-OSMOTIC STRESS:

The cell envelope of gram-negative bacteria including £. coli and S. typhimurium is made up of

two (outer and inner) membranes, with the peptidoglycan cell wall situated just internal to the outer

membrane. The outer membrane has water-filled proteinaceous channels across it that are called porins,

. which allow the diffusion of hydrophilic molecules less than 600 Da. The inner membrane (also called the

2

cytoplasmic membrane) is impermeable to most solutes and acts as an osmotic barrier. The periplasmic

space refers to the aqueous compartment between the inner and outer membranes, and the cytoplasmic

compartment is that which is delimited by the inner membrane.

The response of the cells to an increase in osmolarity of the medium can be considered as

biphasic. The immediate response is caused by the passive movement of water across the membrane

from inside to outside. In this phase the cells undergo distinct changes in morphology, volume, turgor

pressure and metabolism (123, 158, 194, 195). Thus, the cytoplasmic volume decreases, cell turgor

collapses and the cells undergo plasmolysis, ie., a seperation of the cytoplsmic membrane from the cell

wall which is reflected in an increase in light scattering and in optical density of the culture (123).

The second phase of the bacterial response consists of long-term or steady-state adaptive

changes in the cells to the increase in medium osmolarity. In this phase, turgor pressure and cell volume

are restored to levels close to those of cells not osmotically stressed. The adaptive processes in this

phase involve physiological mechanisms that lead to an increase in the intracellular concentrations of

certain solutes so as to restore cell turgor (37).

1.4 COMPATIBLE SOLUTES AND OSMOPROTECTANTS:

An increase in the osmolarity of the growth medium causes an instantaneous efflux of water from

the cell and as a result the cytoplasm loses water until its osmotic potential equals that of the external

medium. There is a consequent reduction in cytoplasmic volume (plasmolysis) and loss of turgor pres­

sure. These changes are associated with inhibition of various metabolic processes like nutrient uptake,

DNA replication and RNA and protein synthesis, etc. Long-term adaptation to hyper-osmotic stress in­

volves increase in the intracellular concentration of certain solutes so that osmotic influx of water occurs

leading to restoration of cell volume and turgor (136, 154, 240).

During osmotic adaptation, accumulation of particular solutes occurs as a result of increased

synthesis and/or by increased active transport. The solutes which so accumulate include the inorganic ion

3

K+ and a small set of organic molecules termed 'compatible solutes'; the latter are so called because,

when present even at high (several hundreds of millimolar) concentrations within the cells, they have little

inhibitorY effect on metabolic processes (22). Compatible solutes that are accumulated by a wide variety .. of organisms in response to hyperosmotic stress can all be classified into a relatively small number of

chemical categories. The most commonly found compatible solutes in bacteria, plants and (lower) ani-

mals are (i) the amino acids glutamate, glutamine, proline, gama-aminobutyrate and alanine; (ii) the

sugars sucrose, trehalose, glycerol, sorbitol, glucose and fructose; (iii) the quarternary amine glycine

betaine; and (iv) N-methylated amino acids like taurine (36, 37).

Another term that is somewhat related to compatible solutes is " osmoprotectant" or "osmopro-

tective solute". Osmoprotectants are defined as substances, which when exogenously provided, at low

(submillimolar) concentrations, can promote the growth of organisms in media of otherwise inhibitory

osmolarity. Osmoprotectants are substances that are actively transported into the cytoplasm of cells

grown at high osmolarity and which either function directly as compatible solutes eg., glycine betaine, L-

proline, or are enzymatically converted into a compatible solute eg., choline which is oxidised in two steps

to glycine betaine (127, 214).

The studies of Cayley et al (25) have shown that in E. coli the following are the major osmolytes

that accumulate in the cytoplasm of cells grown at high osmolarity: K+, glutamate, trehalose, proline and

glycine betaine. The accumulation mechanisms of each of these compounds are further discussed.

1.5 PHYSIOLOGY AND GENETICS OF SOLUTE ACCUMULATION IN E.

coli UNDER OSMOTIC STRESS:

Genetic studies on osmoregulation in E. eo/i and S. typhimurium have revealed that the protein

products of osmoresponsive genes constitute systems for uptake or synthesis of compatible solutes. The

nature of the solutes accumulated in response to osmotic stress and the pathways of their synthesis or

uptake under osmotic stress conditions are discussed in brief below.

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1.5.1 Potassium ion:

K+ ion is the predominant intracellular cation in E. coli. The intracellular concentration of ~ in E.

coli under normal growth conditions (in medium of ~280 mOsm) is around 150 mM (30, 58) and this level

is believed to be required for the optimal activity of many cellular enzymes and for proper DNA-protein

recognition.

The steady-state intracellular concentration of K+ increases from 150 mM to 550 mM upon in­~

crease in the external osmolarity fro in 1 00 to 1200 mOsm, implicating a role for !(+ in maintaining cell

turgor (58).

Intracellular K+ concentration is a result of the equilibrium between K+ efflux and influx systems.

The various K+ transport systems that are involved in uptake of K+, are the high-affinity inducible Kdp

system (59) and low-affinity constitutive TrkA and TrkD sxstems (60, 190). E. coli also possesses two

other K+ transport systems, the KefB and KefC systems, that are involved inK+ efflux (60).

Among the K+ transporters, the Kdp system is most well studied and a role for Kdp in osmoregu-

lation has been implicated (58). The Kdp transporter is encoded by the kdpABC operon and constitutes

membrane-associated uptake system with K+-dependent ATPase activity. Kdp system has a very high

affinity for K+ with a Km of 2 J..LM and Vmax of 150 J..Lmol I g min. Energy for K+ uptake through this

system is provided by ATP hydrolysis. Until recently, cell turgor was thought to be a determinant for kdp

induction; however, this notion of turgor regulation of kdp has been shown to be incorrect, as is further

discussed below.

1.5.2 Glutamate:

The steady-state level of glutamate has been found to increase in conditions of hyperosmolarity

in E. coli and in other bacteria (154, 220). Under these conditions, glutamate also forms the principal

component of the amino acid pool (220). It is presumed that glutamate accumulates in cells subjected to

5

hyperosmotic stress in order to serve the function of a counter-ion to K+, and thus to maintain the elec­

troneutrality of the cytoplasm (192). The observation that accumulation of glutamate occurs even in

bacteria grown in minimal medium (lacking glutamate or glutamine) suggested that this occurs through

enhanced synthesis (220), and Csonka and Hanson (37) have argued that the activities of glutamate

dehydrogenase and glutamate synthase (the two enzymes which independently catalyze glutamate

synthesis) are activated by hyperosmotic stress.

Although glutamate is the predominant anion, it is accumulated only to 50% of the K+ ion concen­

tration in high-osmolar conditions (192). Alkalinization of the cytoplasm due to osmotic stress {220), and

the subsequent efflux of protons (192), has been proposed as a compensatory mechanism to balance the

remaining 50% charge due to increase in [K+]. As an alternative model, Munro et al (168) had suggested

that the excretion of putrescine (a divalent cation) upon increase in medium osmolarity, could be a means

of balancing the charge effects associated with increase inK+.

1.5.3 Trehalose:

Trehalose is a non-reducing disaccharide of glucose ahd, under hyperosmotic conditions,

accumulates to a level equivalent to 20% of the external osmolarity (131). Two genes otsB and otsA,

organized in a single operon, are necessary for the synthesis of trehalose and are induced at the level of

transcription by an increase in medium osmolarity (75). otsA encodes trehalose-6-phosphate synthetase

[whose substrates are glucose-6-phosphate and uridine-di-phosphate (UDP)-glucose] and otsB encodes

trehalose-6-phosphate phosphatase (94). Mutations in otsA or otsB abolish the synthesis of this sugar

and confer an osmosensitive phenotype (75). It has been recently shown that transcription of otsA and

otsB genes depends on the stationary-phase specific sigma factor (RpoS or cf) of RNA polymerase.

Growth-phase-dependent induction of these genes is drastically reduced in mutants lacking this sigma

factor (94).

6

It has also been shown that the accumulation of trehalose can occur only by endogenous synthe­

sis and not by active transport from the medium, even if exogenous trehalose has been provided {20). On

the other hand, exogenous trehalose can be used as C source for growth by E. coli both at low and at

high osmolarity. For the latter purpose, trehalose is transported via a PTS system at low osmolarity,

whereas at high osmolarity it is first hydrolyzed to glucose by a periplasmic trehalase enzyme (whose

expression is specifically stimulated under these conditions) {19, 20). Thus, trehalose is a compatible

solute but cannot serve as an osmoprotectant.

1.5.4 Choline:

Choline can act as an osmoprotectant in E. colithrough an indirect mechanism; when exoge­

nously provided, it is actively transported (through the transport system encoded by betT gene) into the

cytoplasm and is converted to glycine betaine in a two-step oxidation reaction which requires the activi­

ties of the betA- and betB-encoded proteins {6, 127). Thus, choline is an example of a compound which is

osmoprotective but by itself is not a compatible solute. The betA, betB and betT genes are all transcrip­

tionally induced (about 7-10 fold) by an increase in medium osmolarity {62). The betA, betB and betT

genes are located very close to the lac operon (at 7.5 min on the E. coli chromosome map), and

several Alae strains of E. coli (including MC41 00 and its derivatives used in this study) are also deleted

for these genes. S. typhimurium which is naturally deleted for the lac locus, also lacks the bet locus and

therefore cannot be osmoprotected by choline. However, transfer of the bet genes from E. coli confers

choline-mediated osmoprotection in S. typhimurium (6).

1.5.5 Proline and glycine betaine:

L-proline and glycine betaine (N,N,N-trimethyl glycine) are the most commonly accumulated

compatible solutes in a wide variety of bacteria and plants {37, 136, 154, 192, 240). When subjected to

osmotic stress, both E. coli and S. typhimurium accumulate proline and glycine betaine through en­

hanced transport {83, 135, 213). Although proline can both be synthesized for use in protein synthesis

and be metabolized as sole CorN source for growth, neither of these processes contributes to the

physiology of osmoregulation. On the other hand, glycine betaine cannot be metabolized by these organ­

isms (135). Glycine betaine is the most potent of osmoprotectants so far identified for the Enterobacteri-

7

aceae (135). Cayley et al (25, 26) demonstrated that, when proline was used as the osmoprotectant, it

was accumulated to a level nearly equal to that of (residual) cytoplasmic K+; on the other hand, when

glycine betaine was so used, its accumulation was accompanied by replacement of all other osmotically

active solutes (including K+ and trehalose).

There are three transport systems, namely, PutP, ProP and ProU, for the uptake of proline in

both E. coli and S. typhimurium. The PutP system is involved in proline-uptake under conditions where

proline is utilized as sole C or N source for growth, and it is only ProP- and ProU-mediated proline-uptake

that results in osmoprotection (81, 82). Interestingly the same two porters (ProP and ProU) also serve as

the transporters of glycine betaine in these bacteria, and hence play crucial roles in osmotic adaptation

(157). Each of these is further discussed below.

(i) ProP system: The ProP transporter, initially Identified as a proline permease and subsequent­

ly also as a transporter for glycine betaine, is regulated by medium osmolarity at two different levels.

Growth at high osmolarity both induces transcription of proP by about 3- to 5-fold, and enhances transport

activity of the ProP transporter (23, 57, 82). proP mutants are impaired in their ability to be osmoprotected

by both proline and glycine betaine, indicating the importance of ProP in osmotic adaptation.

(ii) ProU system: ProU, encoded by the proU locus, also transports both proline and glycine

betaine under high-osmolarity conditions but appears to possess a higher affinity for glycine betaine than

for proline. Taurine, ectoine, dimethylthetin, etc., which are commonly occurring osmolytes in eukaryotes,

have also been shown to be substrates for ProP and ProU transport systems (28, 112, 153). With the aid

of lac or phoA reporter gene-fusion studies, the proU locus has been independently identified in both E.

coli and S. typhimurium as an osmoresponsive locus, whose transcription is induced more than 1 00-fold

in response to an increase in osmolarity of the medium. !so-osmolar concentrations of both ionic and

nonionic impermeable solutes induce proU to equivalent degrees. On the other hand, glycerol, ethanol

and methanol (which are freely permeable across the cell membrane) do not induce its expression (13,

8

. 24, 57, 81 ), indicating that the signal for proU induction is truly osmotic and not merely a decrea,se in

water activity of the growth medium.

Molecular and genetic analysis of the proU locus indicated that it is an operon with three structur-

al genes, proV, proW and proX(40, 84, 85, 211). All three genes are necessary for exhibition of proU-

mediated osmoprotective effects of both glycine betaine and proline in E. coli (40). The product of proX is

a periplasmic protein which has been shown to bind glycine betaine in vitro (15). The deduced amino acid

sequences of the products of proVand proW show similarities to components of other well-characterized

transport systems such as those for histidine or maltose, permitting the inference that ProU belongs to

the family of A TP-driven binding protein dependent transporters (now called ABC transporters or traffic

ATPases) (5, 96).

1.6 NUCLEOID PROTEINS OF E. coli AND THEIR FUNCTIONS:

The bacterial genome is organized into a highly condensed chromatin-like structure called nu-

cleoid, which also contain several proteins that resemble the eukaryotic histones. The three major "hi-

stone-like proteins" of E. coli, namely HU, IHF and HNS, bind to DNA and are able to compact it (for

review see 55). However, these proteins have not been shown to form complexes (with DNA) that are

analogous to nucleosomes. The role of each of these proteins in the formation of the nucleoid is not

known. Strains that lack one or two of these proteins are viable, but attempts at construction of strains

that lack all the three proteins were not successful (241), suggesting that simultaneous deficiency of all of

them is lethal. Mutations in. genes coding for these "histone-like" proteins have pleiotropic effects on

various facets of recombination, replication, transposition and gene expression.

Mutation in the gene coding for HNS is knowri to affect expression of the osmotically inducible

proU op~ron in £. coli and S. typhimurium (98; 109, 140). In this study mutants deficient in either of the

other two nucleoid proteins, namely HU and IHF, were also shown to be affected in the osmotic regula-

tion of proU. The general features and functions of these proteins are therefore described in detail below.

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1.6.1 NUCLEOID PROTEIN HU:

1.6.1.1 Properties of HU:

HU is a small, basic DNA-binding protein. The two subunits of the heterodimeric HU are HU-c:x

and HU~p, and are encoded by hupA and hupB genes respectively. There are about 30,000 to 50,000

dimers of HU per cell, making it one of the most abundant nucleoid proteins of E. coli. HU binds to both

single- and double-stranded DNA as well as to RNA (101). Introduction of fluorescent labeled HU into

living bacterial cells revealed that HU is primarily associated with the nucleoid and was uniformly distrib­

uted throughout this structure (206), suggesting a role for HU in chromosome organization.

In solution, native HU has an apparent MW of about 20 kDa, which is approximately twice the

MW of the monomers (9.5 kDa). Phosphocellulose chromatography separates purified HU into three frac­

tions. The major fraction amounting to more than 90% of the total protein contains both HU-c:x and HU-p

(196). The two minor fractions which flank the major peak contain only HU-c:x or HU-p (196). Thus it

appears that HU primarily exists as a heterotypic dimer but it can also form both classes of homotypic

dimer. Subsequent biophysical studies have shown that the heterotypic dimer is more stable than either

of the homodimers (175) and that the binding affinity of the various species for DNA follows the order of

HU-c:x,P> HU-P.P> HU-c:x,c:x (217).

HU has the ability to compact DNA as judged both by sedimentation velocity studies (198) and

also by electron microscopic studies of HU-DNA complexes (198). In vitro experiments have ~hown that

HU constrains 14 to 16 negative superhelical turns in SV-40 DNA at a protein:DNA ratio of one (w/w)

(198). Under these conditions, HU-DNA complexes look like beaded-structures with about 275 bp of DNA

bound by 8 to 10 dimers of HU and a linking number change of one per bead.

Though HU has been shown to have no sequence-specificity of binding to DNA, it preferentially

binds to four-way junction molecules (like Holliday junctions) (183) and also to DNA with sharp bends

(called kinked DNA) (183). HU also appears to inhibit cruciform extrusion from supercoiled inverted­

repeat-DNA (183).

10

1.6.1.2 Functions of HU:

Biochemical and genetic studies have implicated roles for HU in the following classes of functions

in E. coli.

(i) Transcription: HU was found to stimulate transcription from bacteriophage Lambda DNA

template by about sixfold at a protein:DNA ratio of one (w/w) (197). Overexpression of HU-cx or HU-~ from

plasmids in hup single mutants or the wild-type strain, leads to repression of the chromosomal hup genes

suggesting autoregulation (124).

(ii) Recombination: HU is involved in various site-specific recombination processes. Transposi­

tion of Mu requires HU protein. Mu transposase forms a stable tetramer complex with the ends of Mu

DNA. In vitro, assembly of this complex was shown to require the HU protein (12). HU has been shown to

stimulate transposition of Tn 10 in vitro (164). Site-specific inversion associated with flagellar phase varia­

tion requires Hin recombinase and factor II. This reaction was also stimulated about tenfold in the

presence of HU (115).

HU has also been shown to affect homologous recombination. In an intrachromosomal recombi­

nation assay, recombination proficiency of strains lacking HU was reduced 10-fold (53).

(iii) Replication: A plasmid system has been developed by Kornberg and co-workers which

contains a chromosomal origin of replication (oriC). HU has been shown to be involved in forming a pre­

priming complex, prior to synthesis of primers and DNA, along with the products of dnaA, dnaB and dnaC

genes. Binding of IHF to the oriC can be modulated by HU; depending upon the relative concentration of

HU and IHF , HU is able to either activate or inhibit the binding of IHF to oriC (18). Strains lacking HU

(hupA hupB strains) were also unable to maintain mini-F and mini-P1 plasm ids (171 r

11

Genetic studies have shown that single mutations in the genes for either HU-a or HU-p have no

. phenotypic consequences. However strains lacking both the subunits are affected in several cellular

functions. Total absence of HU results in reduced growth rate and filamentous cell morphology (8, 105).

Filamentation occurs due to inhibition of cell septation (54). Absence of HU also results in the segrega­

tion of anucleate cells. Surprisingly, overexpression of either subunit also results in cell filamentation

(178). It has been proposed that the lack of HU or imbalanced expression of either subunit interferes with

the cell division process (178).

1.6.2 NUCLEOID PROTEIN IHF:

1.6.2.1 Properties of IHF:

Integration host factor or IHF is the most well studied "histone-like protein" of E. coli. As the name

suggests, it was first identified as a host factor required for site-specific integration of phage Lambda into

the bacterial chromosome during the lysogenization process (160). IHF is a small {22-kDa) basic hetero­

dimeric protein. The subunits of IHF, namely IHF-a and IHF-p, are encoded by himA and hipD genes

mapped at 38 min and 21 min respectively. The strong amino acid sequence homologies between IHF

and HU and the tendency for HU to copurify with IHF and vice versa suggested that the two might share

common features, the ability to compact DNA being one. IHF is also an abundant protein. About 17,000

to 34,000 molecules of IHF monomers are present per cell (47), which is approximately equivalent to

other histone-like protei11s in E. coli. These features of IHF have led to its inclusion in the class of histone­

like proteins of E. coli.

However, unlike other histone-like proteins, IHF is a sequence-specific DNA-binding protein. A

single IHF heterodimer binds to a site of approximately 35 bp that contains a 13-bp consensus sequence,

WATCAANNNNTTR (where W,R and N represent AfT, G/A and AIC/G/T respectively), although the

sequences that flank the consensus are also important. Surprisingly HU seems to enhance the DNA­

binding capacity of IHF to a DNA sequence that does not contain a consensus IHF-binding-site (17).

12

1.6.2.2 Functions of IHF:

IHF affects a variety of cellular functions t~at are physiologically not related in any obvious way

and in all these cases, binding of IHF to the DNA target has proved to be essential for IHF-mediated

effect. However, IHF action is primarily that of an ancillary factor that enhances the binding of various

specific trans-factors to their respective targets. Several studies indicate that binding of IHF induces a

sharp turn at its binding site (176, 180, 186, 225). It appears that all or at least part of the IHF effects on

various systems could be mediated through this change in DNA topology. In vivo and in vitro studies have

indicated roles for IHF in the following functions.

(i) Recombination: The first identification of IHF was that as a factor which greatly stimulates the

lambda site-specific recombination in vitro (121 ). Integration of phage lambda onto the bacterial chromo­

some involves recombination between the phage attP and bacterial attB sites. IHF has been shown to

bind at multiple sites within the attP region. Mutation in one such IHF-binding site, H1, that reduces

binding affinity of IHF to H1, also reduces the frequency of site-specific recombination (73). It is believed

that IHF participates with lnt, another factor required for recombination, in wrapping the·attP site into a

nucleosome-like structure called "intasome" to facilitate DNA transaction. IHF has also been shown to be

required for lysogenization of E. coli by phage P2 (200).

(ii) Packaging of phage DNA: IHF plays an accessory role in lambda DNA packaging by affect­

ing the interaction of the packaging protein 'terminase' with the packaging site 'cos' on lambda (238).

Similarly, the lambdoid phage 21 is unable to grow in himA or hipD mutants. Three potential IHF-binding

sites occur in the phage 21 cos site, and in vitro studies confirm that IHF stimulates phage 21 packaging

(66).

(iii) Transposition: In vivo, a requirement for IHF has been demonstrated for exhibition of the

phenotype of increased transposition frequencies of IS50 and Tn5, in strains that lack Dam methylase

(145). IS50 transposition was increased about 1000-fold and that of Tn5 about 10-fold in Dam- cells. The

mechanisms by which IHF might affect these processes are not known.

13

(iv) Replication: Propagation of plasmid pSC101 has been shown to require the presence of IHF

(21 0). On the other hand propagation of oriC-containing minichromosome requires the presence of IHF

only in strains that are deficient in DNA polymerase I (67).

(v) Gene expression: IHF affects the expression of a large number of genes in E. coli. Analysis

of total cellular proteins in an O'Farrell two-dimensional gel revealed that approximately 15 to 20% of the

polypeptides were different (as judged by intensity or presence of spots), in an IHF mutant strain com­

pared to the pattern in the wild-type parent (70). In vivo experiments suggested that the himA and hipD

genes are negatively autoregulated by IHF (161). IHF mutants were reported to have reduced expression

of genes involved in synthesis of flagellin (216), type I fimbriae (48), isoleucine and valine (176, 177) and

also to have impaired growth on various carbon sources (71, 72). Transcription from the bacteriophage

pl promoter was also stimulated about three- to four-fold by IHF both in vitro and in vivo (76). IHF acts as

a positive regulator of the anaerobically induced threonine deaminase (tdc) operon. In an IHF-deficient

strain, expression from the tdc operon was 10% of that seen in an isogenic wild-type strain (236, 237).

Strains that lack IHF have altered expression and osmotic regulation of OmpF and OmpC, the

outer membrane porins (1 04, 226). In vitro, transcription from ompF and ompC present on linear DNA

fragments was strongly inhibited by IHF (1 04). Mutants lacking IHF exhibited increased expression of

ompF and ompC genes. IHF also negatively regulates transcription of the ompB operon (227) that

encodes the EnvZ and OmpR proteins, the activators of ompFand ompCtranscription. Later experiments

suggested that some of the in vivo effects of IHF on ompC and ompF expression is mediated through its

effect on the ompB regulatory locus (1 04, 186). IHF is also involved in the stationary-phase-induction of

osmYbut not essential for its osmotic induction (128).

IHF can function both as a positive and a negative regulator and in most cases its effect is medi­

ated through modulation of DNA-binding abilities of other trans-factors to their respective cis-elements.

For example: (i) IHF modulates expression of the sodA gene, that encodes manganese-superoxide

14

dismutase, through an enhancement of the ability of Fur and AreA to repress this operon (34). (ii) On a

linear DNA template in vitro, IHF is an essential factor for the transcription from the nitrogen-regulated

and cf4-dependent promoter P2 of the glnHPQ operon. In vitro experiments have indicated that IHF

enhances the ability of NR1 protein to activate transcription from this promoter (32). (iii) In a similar fash­

ion, IHF was shown to be required for Fnr- and NarL-dependent activation of the nitrate reductase (nar

GHJI) operon in response to nitrate availability and anaerobiosis (205).

1.6.3 NUCLEOID PROTEIN HNS:

1.6.3.1 Properties of HNS:

HNS is a small abundant DNA-binding protein which, unlike other histone proteins, is a neutral

protein. HNS acts as a homodimer (subunit molecular weight 15 kDa) and in vitro, binds to both linear

and supercoiled DNA. Binding of HNS to DNA leads to compaction of the latter, as judged by sedimenta­

tion velocity studies. There are about 20,000 copies of HNS molecules per cell. Spassky et al (208)

demonstrated that preparations of HNS protein contain three polypeptides, each having a molecular

weight of 15,500, that are called H1a, H1b and H1c. These authors also suggested that the relative

proportions and the total amount of these three forms vary with growth phase. However, there are other

reports that appear to contradict this observation (see below).

Several workers have shown that HNS preferentially binds to DNA with a native bend in its struc­

ture (239). Recent footprinting studies have shown that at low concentrations, HNS makes sequence­

specific contacts with DNA template, whereas at higher concentrations HNS covers or wraps the entire

DNA template (229). The binding sites for HNS to various promoter regions including lac (193), gal (193)

and proU (141) have been deduced from footprinting studies. These studies have further confirmed the

conclusion that HNS binds to bent-DNA, as the binding sites were primarily tracts of A and T nucleotides

that are known to have high propensity to form a bent-DNA-structure.

(i) hns and its expression: There is a single copy of the hns gene in both E. coli and S. typhimu­

rium, that has been mapped to 27 min and 34 min respectively (182). Expression of hns is controlled both

15

at the transcriptional level and the translational level. The effect of several factors, such as temperature

and growth phase on hns expression, and autoregulation of hns, have been studied by several groups;

some of these results are discussed below.

(A) Autoregulation: Expression of hns is negatively regulated by its own gene product. The

expression of a hns-/acZfusion is 4-fold higher in an hns mutant compared to that in a hns+ background

{43). In vitro, HNS has been shown to act as a repressor for its own transcription, and to exhibit higher

binding affinity for the DNA sequence encompassing the hns promoter region in comparison with that for

a non-specific DNA sequence (231 ).

(B) Growth-phase-dependent regulation: The effect of growth phase on hns expression has

been studied by several groups, but unfortunately their results are not consistent with one another. On the

one hand, Spassky et al {208} had reported an increase in HNS protein from about 15,000 copies per cell

in mid-log phase to about 26,000 copies per cell in stationary phase. Bremer and coworkers {43} also

observed a ten-fold increase in hns-/ac expression (with both gene fusions and operon fusions) upon

entry of cells into stationary phase. On the other hand, Higgins and coworkers {1 00} did not detect any

changes; by Western blot analysis, in HNS concentration as a function of growth phase. Furthermore,

these workers have observed a decrease in expression of about 5-fold from an hns-lux fusion carried on

a plasmid, as cells progressed from mid-log phase to stationary phase {100}.

(C) Effect of environmental factors: HNS has been shown to be part of the cold-shock regulon

of E. coli. Studies using hns-cat and hns-/uxfusions have demonstrated a three- to four-fold induction of

expression upon temperature-shift of cultures from 37°C to 10°C (100, 132}.

In view of the roles implicated for HNS in controlling expression of the osmotically inducible proU

operon, the effect of osmotic stress on hns expression has also been studied. No significant effect of

medium osmolarity on expression of hns was observed for addition of upto 0.3 M NaCI to normal growth

media (100).

16

1.6.3.2 Functions of HNS:

Clues to the functions of HNS have primarily come from studies on hns mutants. Mutations in

hns have pleiotropic effects. The hns locus thus had been identified by several groups working on di-

verse genetic systems, who had each named the locus differently. Thus the locus is now known to be

allelic to (i) osmZ (98), a locus that affects expression of the osmotically inducible proU operon; (ii) bg/Y

(42), a negative regulator of the bgl operon; (iii) drdX (80), that controls the thermoregulation of the pap

fimbriae in E. coli; (iv) pi/G (209), a negative regulator of type I fimbriae in E. coli; and (v) virR (148), a

negative regulator of plasmid-encoded invasion genes in Shigella flexneri. Some of the processes that

are affected by mutation in hns are described below,

(i) DNA rearrangement: Type I pilus expression in E. coli is regulated by the inversion of a 314-

bp segment upstream of, and containing the promoter for, pi/A, the gen.e encoding type I pilin monomer.

In one orientation (ON), this promoter faces the coding region of pi/A, a condition in which pi/A is tran-

scribed; site-specific inversion of this segment results in the promoter facing away from the pi/A gene, the

OFF condition. The frequency of inversion of the "pi/A promoter region increases about 1 00-fold by a

mutation in hns (48).

Mutation in hns leads to a marked increase in the transposition frequency of ·a defective Mu

phage (Mudll 1681) under certain growth conditions (64). Lejune et al (133) had shown that hns mutants

exhibit a 1 00-fold increase in the frequency of spontaneous chromosomal deletion.

In an intrachromosomal recombination assay, recombination proficiency of strains lacking HNS

was reduced by about 100-fold (53).

(ii) Cell division: Strains carrying multiple copies of hns+ gene are known to form long filaments .. containing multiple nucleoids {149). This filamentation was not due to induction of the SOS system,

because introduction of arecA mutation did not alter this phenotype (149). These data suggest that

increased gene dosage of hhs (associated with increased HNS concentration) interferes with cell division

pathway.

17

(iii) Gene expression: The expression of a substantial (yet finite) number of genes is affected by

mutations in hns. Two-dimensional gel analysis of total cellular proteins of E. coli revealed that proteins

· are either strongly induced or repressed in an hns background as compared to their expression pattern in

the hns+ parent {100).

The mutant studies also indicated that HNS negatively regulates expression of the following

genes: (a) the hns gene itself -this autoregulation was more pronounced in log-phase-cells than in sta­

tionary-phase-cells (43, 63); (b) the osmotically inducible proU operon-in this case, hns mutation cause a

partial {3- to 8-fold) derepression of the operon at low osmolarity {41, 98, 100, 140, 174); (c) the rRNA P1

promoter {221); (d) the bgiCSB operon (involved in utilization of p-glucosides)- hns mutation causes

activation of this otherwise cryptic operon {42); and (e) fimA, the gene coding for the phase-variable type I

fimbria( subunit protein of E. coli K-12 {50). Mutation in hns also caused an alteration in the reciprocal

synthesis of the OmpF and OmpC porins {86). In the case of HNS-mediated regulation of hns, proU and

rRNA genes, the protein has been shown to bind to the promoter-proximal region of each of them {221,

229, 231).

It may be noted that HNS does not act as a general (non-specific) repressor of transcription, as

expression from each of several other promoter-lac fusions that were tested was unaltered by mutations

in hns (106). Furthermore, at least in two instances, mutation in hns leads to decreased gene expression:

(a) expression from the promoter P1 of proU operon is reduced at least two-fold in hns mutant strains

{41); and (b) expression of csgA, the gene involved in curli biosynthesis, is reduced two-fold by a muta­

tion in hns {172).

1.6.3.3 Is hns an essential gene?

Hinton et al {1 00) have proposed that the osmZ gene is an essential gene, on the basis of the

observation that all presumed null mutations in osmZ sequenced to date either have been in-frame dele­

tions or have been insertions in the C-terminus which could still express the truncated protein. However,

18

Yasuzawa et al (241) have constructed a null mutation in hns that carry a Hygr gene in the middle of the

hns coding region. Such a mutant strain exhibited phenotype similar to other hns strains, such as ability

to utilize p-glucoside, and also did not produce any detectable polypeptide that cross reacts with anti-HNS

antibody. This strain, however, did not exhibit any altered growth rate or morphology in·nutrient rich

medium.

1.7 STATIONARY PHASE OF GROWTH AND RpoS:

Until some years ago, studies on E. coli physiology and cell metabolism were largely confined to

cultures in logarithmic phase of growth. Recently, however, interest has grown in the physiology of sta­

tionary-phase cells. These developments are briefly reviewed below, since one of t~e major findings

reported in this thesis that the P1 promoter of proU is induced in stationary-phase cells.

1. 7.1 Stationary phase response:

Entry into stationary phase is accompanied by changes in cell shape (ellipsoidal in Jog phase to

spherical in stationary phase), composition of cell envelope (altered membrane composition) and altered

metabolism. Cells in stationary phase are also more tolerant to various stress conditions compared to

cells in logarithmic phase of growth. For example, cells of stationary phase are more thermotolerant

(114), osmotolerant (113) and resistant to high concentrations of H2o2 (113). Only recently the tolerance

to various stress conditions in stationary phase cells are being correlated to presence of certain metabo­

lites and/or proteins, synthesis of which are induced during entry into stationary phase. The changes in

cellular metabolism during entry into stationary phase can be called a stationary-phase-response that

prepares the cell for a prolonged starvation and protects it from various environmental assaults that the

cell may encounter during this period. Some of the genes that are directly or indirectly involved in the

stationary-phase-response have been shown to be growth-phase regulated; expression of these genes is

also dependent on a novel sigma factor RpoS (rf).

1.7.2 RpoS as a central regulator of stationary phase response:

Several genes are known whose expression is induced from two- to fifty-fold during entry into

19

stationary phase. Search for regulators that control the growth phase regulation of these genes has led to

the identification of a locus now designated as rpoS (129). Thus rpoS was discovered in different contexts

and is allelic to nur (230), a mutation that confers sensitivity to near UV irradiation; katF (203), that con­

trols the expression of catalase HPII; and appR (224), involved in expression of acid phosphatase. A

similar gene has been identified in S. typhimurium and found to be essential for expression of virulence

genes needed for survival in macrophages (65).

The rpoS gene has been cloned and sequenced and the predicted amino acid sequence is that

for a 38 kDa protein (165). The amino acid sequence of the rpoS gene product exhibited strong homolo­

gy over its entire length to the family of sigma factors of RNA polymerase (165). Tanaka et al (219) have

purified the rpoS gene product (cf} and have demonstrated that in vitro the reconstituted RNA polymer­

ase (Ecf) has the ability to initiate specific transcription from the promoters of certain genes, thus estab­

lishing the rpoS-encoded protein as a functional sigma factor.

Nucleotide sequence of the rpoS gene has been determined for several diverse £. coli strains

(111). The base sequence as well as the predicted amino acid sequence of the protein exhibit considera­

ble heterogeneity amongst the different strains. Interestingly, Kolter's group has isolated certain rpoS

alleles that confer growth advantage to aged cultures (243). During prolonged incubation, strains carrying

these alleles have a competitive advantage and replace the wild-type population in the stationary-phase

cultures. One of these rpos alleles (rpoS819) bears a 46 bp duplication at the 3'end of the gene, resulting

in a frame shift that replaces the final four amino acid residues with 39 new residues.

Transcription start-sites of several cf controlled genes have beendetermined. However, a

consensus sequence common to the promoter regions of all rpoS controlled genes has not been identi­

fied (92). A putative consensus has been proposed for katF, xthA and bo1Ap1 (16); however, cysA and

osmB have very different promoter sequences from this consensus (3). Several promoters have been

shown to be efficiently recognized by both Ecf and Ed0 in vitro (219); this observation suggests that cf

might recognize a promoter sequence that is not too different from that of d 0 (219).

20

1. 7.3 Regulation of RpoS synthesis:

Expression of rpoS is controlled at the transcriptional and also at the translational level. Studies

using transcriptionaltpoS::IacZfusion have shown that, in rich medium, transition into stationary phase is

accompanied by a five- to ten-fold induction of expression {129, 167). Studies using translational lac

fusion to rpoS, have shown that there is an eight-fold induction of expression in an rpos+" strain during

entry into stationary phase {139}; in a rpoS mutant strain. the fusion protein was expressed at a higher.

level suggesting rpoS autoregulation {204).

Expression of rpoS was shown to be negatively regulated by cAMP {129} and positively regulated

by ppGpp {74). In a.Acya strain, rpoS expression was higher and occured in late exponential phase

{129}. Furthermore, in abcya strain grown in minimal-glucose medium, addition of cAMP repressed rpoS

expression {129}.

1.7.4 Role of RpoS in gene expression:

The number of identified rpoS-regulated genes continue to increase. The role of 1poS and rpoS-

controlled genes are only now beginning to be understood. Several of the genes, expression of which are

dependent on rpoS are involved in the synthesis of protective metabolites, like glycogen {93), trehalose

{75, 94, 119} or degradation of toxic compounds like H2o2 {138}. It is now clear that the gene products

whose expression is induced during entry into stationary phase are involved in pr~~,-Jhe cells for • ,. :"\ ~ - ""'1) .

long-term starvation and conferring general stress resistance. l/ 6 ~ '.. &lbrat, \!.. . ·t l • .,. . " ... ~ .

The large number of genes that are controlled by RpoS include bolA {2, 130 , o sBA (75, 94},

treA {19}, kat£{138}, xthA (201, 202}, osmB (118}, gigS (93}, appCBA (39}, appY(10), mcc (44), dps (4)

and osm Y (128} . Many of these, including otsBA, treA, osmB and osm Y, were first identified as osmoti-

cally regulated genes. Analysis of total proteins on two-dimensional 0' Farrell gels has shown that there

are many proteins (at least 18} that both exhibit osmotic induction and are dependent on RpoS for their

expression (95}. The question that thus arose was whether tpoS has a role in osmotic regulation in addi-

tion to its role in early stationary-phase regulation of gene expression: Hengge-Aronis et al (94, 95) had

suggested, on the basis of data from instantaneous induction assays, th~t several genes including otsBA

exhibit loss of osmotic inducibility in an rpo$ background. Expression of bolA and gigS which were known

to be rpoS dependent, were also shown to be induced upon osmotic upshock and in an instantaneous

induction assay this osmotic induction was abolished in an rpoS strain (95).

Notwithstanding these observations, the role of RpoS in osmotic induction is not clearly estab­

lished. Firstly, Kaasen et al (119) showed that during steady-state growth, osmotic inducibility of otsBA

was not completely abolished in rpoS mutants even though basal expression was considerably reduced.

It is likely that the differences in results obtained by Hengge-Aronis et al merely reflect differences in

assay strategies, and that RpoS itself does not directly mediate osmotic induction of the various genes

under its control (92). Secondly, there does not seem to be any correlation, upon osmotic induction,

between the amount of rpoS gene product and expression of the rpos-controlled genes; the amount of

RpoS did not increase till 90 minute after osmotic shock, and increased only two-fold thereafter (95).

Finally, the level of osmotic induction and of stationary-phase-induction for any given promoter was not

identical, suggesting differential regulation (92).

1.8 REGULATION OF EXPRESSION OF THE OSMORESPONSIVE

GENES:

Expression of most of the ·genes that play a role in osmotic adaptation of the cell are regulated at

the level of transcription, though some are also regulated at the post-transcriptionallevel. Of the several

genes whose transcription is affected by medium osmolarity, it is for the kdp and proU operons and for

the ompC and ompF porin genes that the control mechanisms have been studied in some detail. Genetic

and molecular characterization of the cis-regulatory regions and the trans-factors involved has revealed

the existence of complex regulatory networks in each of these cases, but as yet the primary inducing

signals are not well understood for most of these. The studies reported in this thesis deal with the tran­

scriptional control of the osmotically inducible proU operon, and hence a brief account of what is already

22

known about such control in the cases of other osmotically regulated genes, as also of the proU operon

itself, has been provided below.

1.8.1 Control of ompC and ompF expression:

E. coli cells possess two major outer membrane porins, composed of OmpC and OmpF polypep­

tides respectively, that allow passive diffusion of small hydrophilic molecules. The synthesis of OmpC and

OmpF is controlled by a variety of environmental factors, such as carbon source, growth temperature and

medium osmolarity (14, 86, 142, 233). The total cellular levels of the Ompc and OmpF porins are kept

relatively constant, but depending upon the growth conditions the relative proportion of the two exhibit a

change. The synthesis of OmpC is induced by increased growth temperature and an increase in medium

osmolarity, under which conditions the expression of OmpF is repressed. Synthesis of OmpF and OmpC

are regulated both at the level of transcription and translation and are discussed in brief below.

1.8.1.1 Transcriptional control of OmpF and OmpC synthesis:

The synthesis of OmpF and of OmpC is dependent on the OmpR and EnvZ regulatory proteins.

OmpR and EnvZ are coded by the genes ompR and envZthat together form an operon called ompB (90) .

. EnvZ and OmpR are part of a dual-component sensor-effector system (212) in which the former acts as a

sensor of environmental signals and the latter as an effector that transduces this signal.

The EnvZ protein is an integral-membrane protein with a cytoplasmic domain and a periplasmic

domain (1, 69, 108, 109), and exhibits homology to the family of signal-sensing proteins. The OmpR

protein is a cytoplasmic protein that, upon phosphorylation, activates transcription of the porin genes. In

vitro, the membrane-bound form of EnvZ has the ability not only to phosphorylate OmpR but also to

dephosphorylate OmpR-P, suggesting a role for EnvZ in modulating the phosphorylation status of OmpR

through its kinase/phosphatase activity {223}.

OmpR serves as an activator of ompC and as both an activator and repressor of ompF expres­

sion. The activation, repression and DNA-binding functions can be disrupted independently by mutations

23

in the OmpR protein itself (199). Mutant EnvZ proteins that appear to have lost the kinase activity while

retaining the phosphatase activity, abolish expression of both the porin genes. These results have led

Tsung et al (228) to propose a model in which EnvZ mediates osmotic regulation of ompF and ompC

genes by controlling the concentration of a single species, OmpR-P.

The nucleoid protein IHF has been shown to be a direct negative effector of both the ompF and

ompC operons. In vitro, IHF inhibits transcription from two out of the three ompC promoters (104). This ...

result is consistent with the observed increase in expression of ompC in strains lacking IHF (104). In vitro

IHF also inhibits transcription from ompF. Tsui et al (226) have demonstrated binding of IHF to the pro-

mater region of ompF. The in vitro inhibition of ompFtranscription can be relieved by increasing the

concentration of OmpR in the system, suggesting that IHF may regulate ompFtranscription by altering

OmpR interaction with the ompF regulatory region.

1.8.1.2 Post-transcriptional control of OmpF and OmpC synthesis:

Expression of the ompF and ompC genes is also controlled at the post-transcriptional level.

Mizuno et al (163) have found that a 174-nucleotide-long RNA is synthesized from a promoter that is

situated immediately upstream of, and that initiate transcription in a direction away from, the ompC gene.

This RNA, designated mic RNA, is complementary to the translational-initiation region of ompF mRNA

and was historically the first example of involvement of natural antisense RNA in regulation of gene

expression. Presence of mic RNA gene on a multicopy plasmid completely abolished the synthesis of

the OmpF protein, and this effect of mic RNA was shown to be exerted at the level of translation of OmpF

(163). Deletion studies on the micF promoter region fused to the JacZ gene has revealed a distinct cis-

regulatory region responsible for OmpR-dependent activation of micF expression. This region contains

the OmpR-binding-site, and is responsible for coordinate regulation of both micF and ompC expression in

response to changes in medium osmolarity. Expression from ompC promoter stimulates micF expression

and this is consistent with the model that ompC and ompF are inversely reguiated. However, the single

copy effect of mic RNA on the regulation of OmpF synthesis is negligible, and thus the role of mic RNA in

24

vivo remains unknown {147).1n strains lacking OmpR, expression of micFin cells grown at 24°C is about

1 0-fold less compared to the wild-type, but did not differ significantly from wild-type cells when cells were

grown at 37°C. Furthermore, expression of micF, that lacks the upstream OmpR-binding site, was ther­

moregulated but not osmoregulated, suggesting a role for OmpR in osmotic regulation of micF expression

{35).

The control of OmpF and OmpC thus involve multiple trans-factors including EnvZ, OmpR, IHF

and mic RNA. Though the signal transduction pathway in this system involving EnvZ and OmpR is well

understood, the signal that is sensed by the EnvZ protein remains unknown.

1.8.2 Regulation of kdp expression:

The Kdp transporter is regulated both at the level of gene expression and of transport activity.

The activity of Kdp transport system has been shown to increase with an increase in the osmolarity of the

growth medium at low concentration of K+ ([K+]e); however, a high [K+]e inhibits its transport activity

{191}. The expression of Kdp system in E. coli has been shown to require the products of 2 genes, kdpD

and kdpE which together constitute an operon adjacent to and immediately downstream of the kdpABC

operon {181 ). The KdpD and KdpE protein function as sensor and response-regulator respectively (in yet

another example of the two-component regulatory system) in controlling transcription of the kdpABC

operon. The C-terminal half of KdpD is homologous to sensors EnvZ and PhoR, and KdpE is homologous

to effectors such as OmpR and PhoB.

The expression of kdp is induced by low [K+le (conditions at which the constitutive Trk systems

forK+ uptake are ineffective) and is repressed by high [K+le (when the Trk systems are functional). Using

a kdp-Jac operon fusion strain, Epstein and coworkers {126} demonstrated that kdp induction begins at

that [K+le where concentration of K+ becomes limiting for.growth. However, the exact nature of the signal

regulating kdp expression has remained elusive.

25

1.8.2.1 Model advocating turgor as a signal:

Laimins et al (126) had cited the following observations to suggest that kdp expression is regulat­

ed by the osmolarity of the growth medium. (i) the threshold value of [K+]e below which the steady state

kdp expression was induced and above which it was repressed, was determined by and was directly

proportional to the concentration of NaCI present in the growth medium. (ii) An osmotic upshock caused

by addition of ionic or nonionic impermeable solutes to cultures grown at a [K+]e sufficient to repress kdp,

caused an instantaneous transient increase in transcription of kdp. Glycerol which is freely permeable

across the cell membrane, did not cause this transient induction. Based on these results, Laimins et al

(126) proposed that turgor pressure is the signal regulating kdp expression and that growth in K+-limiting

conditions imposes a turgor stress on the cells. The turgor-regulation model of kdp expression has been

widely accepted by workers in the field of osmoregulation, and this system is considered to be the para­

digm in biology for control of gene expression by mechanical signals or stimuli.

1.8.2.2 Model advocating transmembrane K+ flux as a signal:

In a recent report, however, Asha and Gowrishankar (9) have reexamined the turgor regulation

model for kdp by analyzing kdp-lac expression in various genetic backgrounds and under varying growth

conditions.

Based on the following arguments these workers have suggested that the signal for kdp induction

cannot be turgor. (i) During steady-state growth, kdp expression was induced by several ionic imperme­

able solutes to different extents whereas non-ionic impermeable solutes had no effect on kdp expression;

(ii) during growth under high osmolar conditions, accumulation of compatible solutes such as trehalose or

glycine betaine, that are expected to restore cell turgor, had no effect on kdp expression; and (iii) a muta­

tion in the kdpDE regulatory locus, that resulted in altered signal perception, provided a clue to the fact

that even in the range between 2 and 30 mM [K+le (conditions in which cell turgor is expected to be

maximal and remain constant), the signal regulating kdp did not remain constant. As an alternative model,

these workers have proposed that the specific rate of transmembrane K+ influx serves as a signal for kdp

26

induction, such that a decrease in flux activates the kdp operon. However, for such an mechanism to

work, the flux values forK+ import through the various K+ porters must be integrated so as to provide a

single value of signal strength, and the candidate for such an integrator remain hypothetical {9}.

1.8.3 Regulation of proU expression:

Transcription of proU is induced several hundred-fold upon growth in high-osmolarity media {24,

57, 81, 89, 150), making this quantitatively the most prominent example where a mechanical stimulus

controls gene expression. However, the mechanism of proU regulation is as yet poorly understood, and

no trans-mutant has been obtained in which osmotic regulation of proU is completely abolished {56}.

Studies in E. coli on the cis-regulation of proU have shown the presence of two promoters P1 and

P2, with the sequences immediately around each conferring five- and eight-fold osmotic inducibility on

transcription initiated from the respective promoters {41, 84, 173, 174). A negative regulatory element

residing in the sequence downstream of P2 (within the first strl!ctural gene proV) is also necessary for

repression of proU expression at low-osmolarity ( 41 , 173, 17 4). The three mechanisms described above

appear to contribute additively to proU osmoresponsivity.

The results of recent studies in S. typhimurium by the groups of Csonka {173} and of Higgins

{174} have been consistent with the conclusions for P2 promoter and NRE above, although this organism

appears to lack the promoter P1. The effect of the NRE appears to be specific for proU transcription,

because its placement downstream of heterologous promoters does not confer osmoresponsivity of

transcription (41, 173}. Mellies et al (155) have described a salt-resistant transcription component of a

'minimal' promoter which may be identical to the P2R mechanism defined herein. However, factors that

mediate osmotic regulation of the promoters P1 and P2 have not been identified.

Based upon the physiological and genetic data on proU regulation, two different groups have

proposed alternative models for proU regulation which invoke changes, respectively, in DNA topology or

in intracellular ionic environment as the factors that mediate osmotic induction of proU expression. Hig-

27

gins and coworkers (98) have suggested that changes in DNA supercoiling play a primary role in the

osmotic induction of proU. In an alternative model, Villarejo and colleagues (185, 187) have postulated a

key role for potassium glutamate in directly activating the transcription of proU. The experimental support

for each of these models.is discussed in brief below. It may also be mentioned here that neither model

has yet been convincingly demonstrated to be true.

1.8.3.1 Model advocating supercoiling as the signal for proU expression:

In the model proposed by Higgins and coworkers (98), increase in proU expression at high

osmolarity is determined by an increase in (negative) superhelical density of DNA. In support of this

model, Higgins et al (98) cited the following observations. (i) Strains carrying mutation in topA, the gene

encoding topoisomerase I, exhibited increase in negative supercoiling and also exhibited increased proU

expression; (ii) treatment with the drugs nalidixic acid and novobiocin, inhibitors of DNA gyrase activity,

reduced negative supercoiling and also reduced osmotic inducibility of proU expression; (iii) growth of

cells in medium of high osmolarity, a condition in which proU expression is induced, caused an increase

in n~gative supercoiling; addition of glycine betaine, to the high-osmolarity medium (that leads to restora­

tion of cell turgor) resulted in both reduction in proU expression and restoration of supercoiling to the level

characteristic of that in cells grown at low osmolarity; (iv) a category of mutants in a new locus designat­

ed osmZ, derepressed proU expression at low osmolarity. Plasmids isolated from these mutant strains

also exhibited increased negative supercoiling at low osmolarity. Subsequently the osmZ mutation was

shown to be allelic to hns (80, 103, 106, 120, 149), the gene encoding the nucleoid protein HNS; and (v)

anaerobicity, a stimulus known to increase the global negative supercoiling of DNA also affected maximal

induction of proU expression under high-osmolar growth conditions (169). Together these results led

Higgins and his colleagues to propose that proU transcription is regulated primarily by environmentally

induced changes in DNA supercoiling.

In demonstrating this correlation these workers have employed changes in linking number (5Lk)

of reporter plasmids as a measure of superhelical density changes in the cell. However, several of the

28

associations reported between 8Lk variations and particular mutations (notably hns) or environmental

conditions (notably high osmolarity) might be reflective of alterations in constrained DNA supercoiling

rather than a change in DNA superhelicity. Thus, although changes in supercoiling do affect proU expres­

sion, whether osmotic induction of proU expression is mediated by changes in DNA superhelicity remains

questionable.

1.8.3.2 Model advocating potassium glutamate as a signal molecule controlling

proU expression:

The supercoiling model has been challenged by Villarejo and coworkers (185, 187), who have

instead proposed an alternative model which suggests thatpotassium glutamate acts as the molecular

signal to stimulate proU expression. This model is based on the following observations. (i) In a reconsti­

tuted cell-free coupled transcription-translation system (S-30 extract), the addition of 0.1 to 0.3 M potassi­

um glutamate was shown to specifically stimulate expression of proU in a concentration-dependent

manner; potassium acetate was a relatively weak stimulator, and other ionic or non-ionic solutes totally

lacked stimulatory activity (117, 187). (ii) The specificity of potassium glutamate as an inducer of proU

transcription has also been demonstrated in a simple in vitro transcription system (185). Transcription

from the proU promoter using purified components such as plasmid DNA template, cr70-RNA polymer­

ase holoenzyme and the four ribonucleotides, in the absence of any other protein factor (except an

RNAse inhibitor) was shown to be strongly stimulated by the addition of 0.3 M potassium glutamate;

transcription from promoters of genes that are not osmotically regulated eg., bla, pepN and lac was inhib­

ited under these conditions. Taken together with the fact that intracellular potassium glutamate concen­

tration during steady-state growth increases with increasing osmolarity of the growth medium {25, 26, 61,

215), the data suggested that osmotic control of proUtranscription is mediated by direct action of potas­

. sium glutamate on the transcription complex.

Jovanovich et al (117) also reported reconstitution of proU expression in a coupled transcription­

translation system in vitro, and demonstrated that the optimum potassium glutamate concentration for

maximal expression of proU (0.3M) was higher thari that for the activity of /acUV5 promoter(0.1 M). These

29

workers had also implicated a role for a stable, nondialyzable macromolecular factor, that is present only

in cells grown in high osmolar media, in osmotic regulation of proU expression, but this finding has not

been replicated (187).

The model for potassium glutamate being involved in proU regulation was also supported by the

following observations of Sutherland et al (215). (i) Introduction of a mutation in the kdp locus, or growth

in potassium-limiting medium, led to reduced levels of intracellular potassium and to a decrease in

osmotic inducibility of the proU operon; expression of other operons, and total protein synthesis, was

relatively unaffected under these conditions. (ii) The glycine-betaine mediated decrease in expression of

proU-/ac in high-osmolarity medium exhibited good correlation with the decrease in intracellular potassi-. urn concentration under these conditions.

Although the genetic and biochemical studies suggest a role for K+ in optimal expression of the

proU operon, the observed magnitude of proU induction in vitro was far less than that observed in vivo.

Thus it is possible that although the expression of proU is impaired under K+ -limiting conditions, other

factors are also required for full osmotic induction of proU expression.

1.9 OBJECTIVE IN UNDERTAKING THE PRESENT STUDY ON OSMOT­

IC REGULATION OF proU OPERON IN E. coli:

As discussed above, theproU operon in E. coli encodes an osmotically inducible uptake system

for proline and glycine betaine which plays an important role in osmoregulation. The structure of the

operon, and the cis-mechanisms involved in proU regulation have been earlier characterized. At least

three distinct cis-mechanisms (referred to in this study as P1 R, P2R and NRE respectively) appear to

contribute additively to proU osmoresponsivity: (i) sequences around promoter P1 which is situated 250

bp upstream of the first structural gene pro V; (ii) sequences around the second promoter P2 which is

situated 60 bp upstream of proV; and (iii) a negative regulatory element (NRE) present within the coding

region of pro V, deletion of which results in derepression of proU expression at low osmolarity (Fig. 1.1 ).

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P1 regulation (P1 R, 6-fold)

P1

I I I I I I I I I

-

P2

P2 regulation (P2R, 8-fold) ~ Negative regulatory element (NRE, 25-fold)

I I I I I

proV

Fig. 1.1. The three cis elements in E. coli proU regulation. The relevant regulatory region of the proU operon is represented to scale by the top horizontal line. The transcription initiation sites for the promoters P1 and P2 are marked by arrows, and the proV coding region by solid box. The transcription initiation sites of P1 and P2 are 250 and 60 bp upstream of translation initiation site of proV respectively. The cis elements P1 R, P2R and NRE are contained within the demarkated sequence region shown against each, and their individual contribution to proU osmotic inducibility are indicated.

Genetic and biochemical studies have, however, failed to identify any classical regulatory protein

(s) involved in osmotic regulation of proU expression. The major models of proU regulation thus have

invoked non-protein factors like K+ ions and DNA-supercoiling as modulators of proU transcription. Al­

though experimental data exist to partially support each of these models, neither model has proved to be

satisfactory in providing a bomplete explanation for osmotic regulation of proU expression.

If the complex multiple cis-mechanisms involved in proU expression is any indication of the

possible complex multifactorial trans-regulation of proU, then it is easy to explain the failure to obtain

single gene mutations that completely abolish proU osmotic induction.

In this study, fresh attempts were made to isolate mutations that would affect proU expression in

trans, and to study the effect of each of the mutations on the three cis-mechanisms described earlier. In

this way, one may be able to identify factor(s) that have a profound effect on one or the other cis regulato­

ry mode, even when their effect on the native proU operon may only be marginal.

Using the approach outlined above, several mutations were identified that affected proU expres­

sion in trans, The results of these studies are presented in three chapters. Chapter 3 describes the isola­

tion and characterization of mutants that are affected in proU expression. The effects of mutations in

various "histone-like proteins" of E. coli on proU regulation are described in this chapter. Chapter 4 deals

with studies on a mutation that specifically affected promoter P1 expression, which established that P1 is

an RpoS-dependent promoter. In the last chapter (chapter 5) the conclusions drawn from the present

studies are collated and compared against existing models of proU regulation.

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