Bacterial shock responses

Bacterial shock responses J. Gareth Morris Bacteria, very early in their history, developedprotective ‘shock responses’,triggered when the organism was exposed to some acute environmental stress.At the heart of each responsewas the accelerated synthesis of both general and stimulus-specified intracellular proteins. So successful was this strategy that its basic mechanism and severalof its componentswere highly conservedthroughout the course of evolution. This was particularly true of heat shock proteins whose universal distribution has both good and bad consequencesfor man. Bacteria (prokaryotes) are the most versatile of living organisms. Evolutionary experimentation threw up species that could successfully inhabit environments that are uncongenial to other forms of life, so that among contemporary specieswe find bacteria that can thrive at higher temperatures than most living creaturescan endure, and others that can grow in media so saline, alkaline or acidic that competitors are virtually non existent. These ‘extremophiles’ startle us by their easy occupancy of environments so hostile that we would have presumed them to be totally sterile. Yet even more ordinary, bacteria are surprisingly tolerant of quite major perturbations in their environments. They react to altered environmental conditions by suppressing certain metabolic activities and expressing others that were previously latent. The rate and duration of the environmental change can determine the nature of the response. Gradual change is more easily accommodated, in that it allows the organism time to ensure that its progeny are fully adapted to the novel circumstance(s)that they will encounter. Abrupt change is the cause of stressor shock, and the primary response of the organism is directed to the conservation of its viability whilst it mobilizes its defences to the specific threat and gains time in which to effect the necessaryadaptation. In this article we shall chiefly be concerned with such stressresponsesas those that are engenderedby rapid temperature change, abrupt nutrient-deprivation or encounter with a potentially toxic chemical. These are not unusual occurrencesfor bacteria in their natural situations, though in most laboratory-based studies it has been the habit of bacteriologists to devise the most appropriately nutritious media, to ex-

J. Gareth

Morris,

D.Phil., F.I.Biol.,

F.R.S.

Is Professor of Microbiology in the University of Wales, Aberystwyth. His research interests are in microbial physiology; in particular anaerobic bacteria and the biotransformations that they can accomplish.

Endowour.

0i60-9327~

New Series, Volum

2

17, No. 1.1993.

tam + 0.00.

Q 1993 persamon

Rerr

Ltd. Printed

in Great Britain.

elude all competitor organisms and to incubate the resultant monoculture in an optimal atmosphere and at an optimal temperatureto ensure that the very quickest rate of growth is sustained for the longest possible period of time. Yet in nature, far from living in perpetual luxury the bacterium might live a precarious existence under near-starvation conditions, grazed on by predators and threatened by competitors. The famous Russian microbiologist S. N. Winogradsky when surveying soil bacteria perceived that they could be divided into two distinct groups: (i) the zymogenous bacteria (now called copiotrophs) which led a spectacular feast and famine life style, and (ii) the less spectacularbut very successful authochthonous bacteria (oligotrophs) which had a ‘fast and famine’ existence but which were content with lower nutrient concentrations even though they did not achieve such dense populations or rapid rates of growth. Despite, or perhaps becauseof, their different growth strategies, both types of organism coexisted very successfully; neither, however, was immune to episodesof stress. In studying bacteria under stressit is almost inevitably Escherichiu coli (E. coli) and Salmonella typhimurium (S. typhimurium) that have been the prime subjects of investigation [l]. This is not only becauseboth organisms can be grown at relatively rapid rates in defined minimal media, but also because they are very amenableto molecular genetic procedures. Furthermore, probably more is known of their physiology during steady state growth than has been established for any other species. In certain genera of bacteria, for example Bacillus or Clostridium, impending starvation or some equivalent stress can trigger the start of a temporally ordered sequence of cellular changes (intracellular differentiation) that leads to the creation of a dormant endospore.This spore is apparently devoid of metabolic activity and is highly resistant to a wide range of potentially lethal agentsincluding heat, desiccation, ultra violet irradiation and exposure to various toxic chemicals. Restitution of suitable growth conditions induces its germination, resulting in the emergenceof a

vegetative cell capable of normal metabolism, growth and reproduction. In contrast, many of the indigenous marine bacteria in the deep oceanslive almost perpetually in conditions of gross nutrient deprivation. Many are able to survive very long periods of starvation despite the fact that they do not sporulate. Instead they appear to undergo a more subtle form of differentiation into vegetative but exceedingly slow growing ultramicro cells [2]. Such behaviour highlights one of the major difficulties that besets all experimental studies of bacterial survival under imposed stress, namely that the bacterial population under examination is heterogeneous in its composition. This is as true of the monoculture grown at a fixed rate in a continuous flow chemostat as it is of the batch culture whose composition visibly changes with time. Subpopulations with distinctive physiological characteristicsare present in relative proportions that are likely to change in line with variations in the mean growth rate of the culture. This has madeit very difficult to assessthe true responseof a bacterial population even to a single environmental stress.Fortunately, proceduresare now being developed - for instance, analytical flow cytometry - that enable measurementsto be made of key physiological properties of single organisms in such non-homogeneous populations. Even so, it remains difficult to relate laboratory-based studies of monocultures to the behaviour of bacteria in their natural situations since therein they generally live as membersof mixed microbial communities. Companion organisms might prove a sourceof stress:for example,by acidifying the environment or by secreting toxic metabolites.On the other hand, neighbouring organisms could act protectively, as in the caseof facuhative aerobeswhose efficient scavenging of oxygen often enables obligately anaerobic bacteria to coexist in a stable consortium that is exposed to air. Yet in whatever situation they find themselves, all bacteria are liable to be subjected to sudden shocks imposed by rapid changesin their environment and they are by no meanspassive in their responsesto such insults. The challenge posedby an abruptly imposed stressis well-described as a shock,

SlbXllUS

Figure 1

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seusor

The circuit

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of cellular

signal

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recognition

for it is evidently so registeredby the subject bacteria. Whatever its nature, it evinces a responsewhose most immediate purpose appearsto be the rapid shutdown of non vital activities, coupled with attempted protection of the most vulnerable structural, genetic and enzymatic elements within the cell and enhancementof the activities’of enzymesdevoted to the repair of any damagesuffered by these components. Taken together, thesereactions provide the organism with the opportunity to kcruit the metabolic machinery that could serve to abateor better tolerate the actual change that has occurred in its environment. The core shock responsemight thus be considered to constitute an automatous reaction to suddenly imposed stress,irrespective of its actual character. The accompanying specific stress responses are directed to combatting the distinctively precise threat that the actual changeposes. It follows that bacteria are sensate organisms to the extent that they are evidently capable of monitoring their environment and of registering changesin its chemical and physical properties that would be likely to affect them for good or ill. The appropriate signal(s) must then be despatchedfrom the sensorsto those effectors that elicit the appropriateresponses.In the event, these effecters are usually regulators of the transcription of genes which encodeproteins whose enhancedor diminished production constitutes the concerted response.The circuit of intracellular communication that links a perceived stimulus to a behavioural response is outlined in figure 1, which also indicates the possibility of feed back attenuation or amplification of the signal. The transcriptional regulator may control the expression of groups of genes and operons distributed around the bacterial chromosome; being subject to this common regulatory control, these constitute a single regulon. The entire set of genes responsive (either positively or negatively) to an identified environmental stimulus constitutes a stimdon. A stimulon is usually comprised of several regulons. Although this network of controls might at first sight appear over elaborate,it servesto concert the activities of a variety of disparategeneswhich under other circumstances might need to be expressedindependently of each other. The most obvious demonstration of environment sensing directed to a purposive response is provided by motile bacteria

Regulator

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and response

Response-

to an external

Bebmiour

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that swim to or from certain chemicals (chemotaxis) or sources of light (phototaxis) [3, 41. Movement along the gradient of chemical concentration is the result of constant monitoring of this concentration by a cell membrane-located sensing protein which, via a sequenceof intracellular proteins, conveys the requisite signal to the flagellar motor which, depending on the direction of bacterial migration, duly extends or shortens the duration of its counter-clockwise rotation [.5]. Strains of E. coli can demonstratechemotaxis. They can seekout a zone of neutral pH in preferenceto alkaline or acidic conditions, can move toward the warmer temperaturein a range of 20-37°C and can demonstrate negative phototaxis by swimming away from a source of blue light. That the more covert stress reactions of bacteria are equally well coordinated can be illustrated by examining the ways in which they react to abrupt increasesin: (a) temperature, (b) oxygen tension and (c) nutrient deprivation. Heat-shock

response

In 1962 F. Ritossa [6] reported that when larvae of the small fly Drosophila bucksii were raised in temperaturefrom 2%30°C and held there for only half an hour, some of the geneson the giant chromosomepresent in their salivary gland cells were activated whilst others were inactivated. On each occasion that the experiment was repeated the samepattern of selectively enhanced and repressedgene expression was visibly evidencedby the appearanceor disappearanceof so-called puffs (manifestations of localized unwinding of the chromosomal DNA). Some 12 years later, the products of the heat-shock activated genes were identified as heat-shock proteins (HSPs) and severalwere purified. The increased initiation of transcription of a set of heat-shockgenesleading to the production of HSPs is now recognized to be a virtually universal cellular responseto an up-shift in temperature. Not only the general mechanism of the heat-shock responsebut also many of its actual components have been highly conserved throughout the course of evolution, with HSPs evidently performing important roles in the physiology of all living cells whether these are prokaryotic, archaeal or eukaryotic [7]. Indeed, basal quantities of several HSPs manifestly play a part in the

normal (steady state) metabolism, reproduction, development and differentiation of cells in higher plants and animals. The magnitude of their transiently increased production in responseto a heat shock is, in part, determined by the magnitude and duration of the temperatureelevation, with pre-exposureto mild heat stressenhancing the resistance of the cell to a subsequent more grave heat shock. When E. coli which has been growing at, let us say, 28°C is abruptly shifted to a higher temperaturefor example 42°C there is a quite dramatic but impermanent increase in the rate of synthesis of at least two dozen of its intracellular proteins. Some 20 of these HSPs are specified by genes whose transcriptional expression is regulated by the protein product of gene rpoH which servesas a sigma factor (Oar) to confer novel specificity on the transcriptional enzyme RNA polymerase. During steady state culture of E. coli its normal RNA polymeraseholoenzyme (HoTo) consists of a core enzyme united with sigma factor 70, and this enzyme complex recognizes those promoters of genes and operons concerned with the housekeeping functions that are essential to vegetative growth and reproduction. When a70 is displaced by the heat shock response-eliciting 032,the changedholoenzyme newly recognizes the heat-shock specific promoters of HSP-specifying genes.Thus 032 serves as the positive regulator of the major regulon in the heat-shock stimulon of this bacterium. There is some suggestion that an ancillary regulon in the samestimulon may be positively regulated by a distinctive 024. Of the major HSPs of E. coli the most prominent are encodedby the dnaK, dnaJ, grpE, groEL and groES genes, and although their rate of production is stimulated by heat shock, they are formed in basal concentrations in cells that are not heat stressed.The DnaK, DnaJ and GroEL proteins act successively in the process whereby newly formed polypeptides are protected against misfolding or aberrant aggregation and are easedalong that route of protein folding which yields the structure with the desired biological activity [8]. In this role these HSPs are serving as chaperonesand in heat-shockedcells they may be expected not only to protect key proteins to which they bind, but also to aid the recovery of temporarily denaturedproteins. Initially however the dnaK, dnaJ and grpE genes of E. coli were identified as performing essential host cell functions in the intracellular replication of bacteriophage h and it is evident that HSPs can perform functions additional to those normally associated with chaperones. Thus some of the E. coli HSPs are capable of proteolyzing irreversibly damaged polypeptides and they have been implicated in processes as diverse as nucleic acid synthesis, cell division and motility, the last via a role in the synthesis of the flagellum.

3

Although positively activated by 032, the heat-shock responseof E. coli is also autoregulatedby its immediate products. A plausible hypothesis supposesthat under steady-stategrowth conditions such 032 as is available is sequesteredby attachmentto DnaK which even at its basal levels exceeds in concentration the lo-30 molecules of 032 that are present per cell (growing at 37°C). Furthermore, free 032 is one of the most unstable of E. coli proteins with a half life in vivo of less than one minute. When the cell is heat-shocked, however, the available DnaK is called upon to bind with heat-damagedpolypeptides, leaving the 032 free to associatewith core RNA polymerase and so institute accelerated transcription from heat-shock promoters.As the damagedproteins are restored to normality, DnaK is again made available to bind 032 with resultant deceleration of heat-shock gene transcription. By this means,DnaK would act as a negative regulator of HSP production and would indirectly serve as a cellular thermometer [9] (though it might share this role with the ribosomal rRNA translational machinery). Interestingly, cold shocking of E. coli (for example reduction of its temperature from 37°C to 1O’C) restricts its growth by interfering with an early step in protein synthesis. Although several novel proteins are produced there is inactivation of 03~ with consequentrepression of synthesis of HSPs. The several protective functions undertaken by HSPs should makeenhancedsynthesis of some of these proteins as helpful to the cell when it suffers other environmental insults as when it experiences a heat-shock. It is not surprising therefore that subsetsof HSPs are subordinately synthesized alongside more specifically directed defensive or repair proteins in E.coZi subjected to oxidative shock, osmotic stressor starvation. Yet, apart from an upshift in temperature, only exposure to ethanol or to certain agentsof nucleic acid damage are known to elicit the complete repertoire of the heat-shockresponse. It has been mentioned that the heatshock response,the manner of its regulation and indeed the structures of some of its components have been evolutionarily conserved. In eukaryotes the positively acting transcriptional regulator is the heatshock factor (HSF) which by binding to specific chromosomal DNA sequences called heat-shockelements(HSEs) enables these to facilitate transcription of downstreamheat-shock genes.The synthesis of families of HSPs is thereby enhanced,for convenience each family being distinguished by the approximate relative molecular masses (in kDa) of their member proteins. Thus the HSPs 70 show about 50 per cent amino acid sequenceidentity with the analogous DnaK protein of E. coli. Structural features of the GroEL of E. coli are highly conservedin the HSP60 chaperones of eukaryotic cells. This close structural resemblance has led to the 4

proposition that certain HSPs could contribute to the development of autoimmune diseasestatesin animals and man [lo-121. The HSPs of most invasive pathogenic bacteria are major antigens and hence immune targets for both B-cell and T-cell lymphocytes, while viral infections can stimulate host cell HSP synthesis. For example, HSP65 is produced both in human cells and as a major virulence antigen of Mycobacterium tuberculosis, the bacterial and human proteins having in common at least four sequences of about 10 amino acids each. Antibodies against HSP65 are abundant in patients with rheumatoid arthritis and it is not inconceivable that an immune recognition system that was evolved to counter infection may under certain circumstances confusedly be deployed against self antigens. In that case the striking conservation of structures of HSP molecules has not been of unalloyed benefit.

proteins whose synthesis is induced by exposure of E. coli to a non-lethal concentration of H202. The intracellular concentration of the OxyR protein is not increased as a consequenceof E. coli encountering H202 and it is presumed that while it is normally in an inactive (presumably reduced) condition it is converted into an active state by oxidation. OxyR thus acts both as sensor and transducer of the oxidative stress signal conveyed by H,O, formation, for in its active state it then binds specifically to promoter sites of the genes whose expression it regulates. The regulation can be negative, as in the case of the autoregulative suppression of its own (OxyR) production, but it may also be positive, leading to accelerated synthesis of the products of genes such as katG (specifying a catalase), ahpCF (encoding an alkylhydroperoxide reductase)and gorA (specifying a glutathione reductase). The SoxRS regulon [ 161 is only one component of the 0,’ stimulon, for in E. Oxidative stress coli over 30 proteins (Soi proteins) are inOxygen in excessis toxic to all living or- ducibly synthesized in responseto its enganisms,yet even bacteria differ greatly in countering a generatorof 01’ anions. The what concentration of oxygen they per- means of regulation of expression of the ceive to be life-threatening, with strict SoxRS regulon differs from that of the anaerobesbeing intolerant of oxygen ten- OxyR regulon in that a two component sions that are routinely enjoyed by aer- sensor-regulator system is implicated. In obes. The responsesto excessoxygen that some undisclosed fashion the O,-’ anion are displayed by bacteria are also very di- causes specific redox activation of the verse. Motile organisms might try to flee SoxR protein which in its active form its presence by displaying negative aero- binds to the promoter of the SOXSgene and taxis, while some non motile bacteria so provokes its accelerated transcription. might seekto escapeits effects by associ- The resultant heightened concentration of ating with oxygen scavenging species.The SoxS protein, either alone or in concert threat seemsto be posed not so much by with active SoxR, switches on transcripdioxygen itself but by products of its par- tion of the SoxRS regulon genes,which intial reduction during the course of its bio- clude genes specifying a scavenging logical utilization. Hydrogen peroxide manganesesuperoxide dismutaseand a reH202 and superoxide anion (the free radi- pair DNA endonuclease. It is somewhat cal, 02-’ are chief among such by-prod- surprising that the responses engendered ucts, acting not only in their own right but by H202 shock and by superoxide stress as precursors of the more damaging hy- are so distinctive. Even though there is droxyl free radical. Living cells that per- some overlap between the two stimulons petually, or in any way usually, encounter and between these and other stress redioxygen, possess means of protecting sponses(figure 2) even the differences bethemselvesagainst the consequencesof in- tween these serve to emphasise the fact tracellular formation of such potentially lethal agents. These measuresinclude the synthesis of enzymes that destroy the primary agents (for example hydroperoxidasesand superoxide dismutases)and the enhanced production of repair enzymes (especially various nucleaseswhich repair single strand-breaks in genomic DNA). Yet it is probably not dioxygen whose concentration is monitored by the bacteria but rather the oxidative stressattributable to its presencein excess [13]. Thus synthesis of someof the componentsof the responseto oxygen stresscan be induced by other oxidants. Indeed, two of the major regulons that comprise the oxidative stressstimulon Figure 2 Bacterial responses to of E. coli (identified by the gene products oxidative stress and possible overlaps that regulate their expression) are (i) the with other stress responses. The OxyR regulon and (ii) the SoxRS regulon numbers in parentheses indicate the numbers of proteins and genes known to [141. be involved in the response. (After Farr The products of the OxyR regulon [15] and Kogoma 1131, reproduced courtesy of are a subset of the 30 or so intracellular American Society for Microbiology.)

that E. cob evidently perceives HZ02 and 02-’ anion as posing non identical threats. For example, transient accelerated synthesis of the chaperone proteins GroEL and GroES is induced in E. coli both by H,O, and by O,-‘, yet production of DnaK, though triggered by H,O, is not induced by O,-‘. Deletion from the E. coli chromosome of the rpoH gene (which specifies 032) not only makes the cells so sensitive to thermal stress that they are unable to grow at temperatures above 2O”C, but it also enhances their sensitivities to both H202 and O,-‘. Furthermore, exposure of E. coli to H,02 but not to a 01’ generator will induce the synthesis of the RecA protein which is crucial to the activation of the so-called SOS response to chromosomal damage. Starvation stress A sudden ‘shift down’ from nutrient-rich to nutrient-poor environments can provoke a distinctive response in even those bacteria such as E. coli which are not able to produce dormant spores [ 171. The purpose served by this starvation response seems to be that of protection of essential cellular components and of viability during a period of adjustment of metabolism to the changed conditions. This period could, for example, see the preferential synthesis of transport proteins whose heightened substrate affinities allow the organism the better to make use of the remaining low concentrations of key nutrients. In E. coli more than 55 starvation-induced proteins (Sti proteins) have been identified. Not all are simultaneously synthesized, for some are produced very early and transiently after imposition of the starvation shock while others appear later but persist longer. The chaperone HSPs DnaK and GroEL are found amongst the Sti proteins and a subset of about 15 of these proteins are induced by a ‘general’ starvation stimulus: that is, independently of whether the starvation is for carbon, nitrogen or phosphate. Other Sti proteins form a set that is uniquely induced by carbon starvation, others are specifically induced by nitrogen deprivation and still others by phosphate starvation. Overlap of these stimulons leads to the existence of non identical subsets of Sti proteins whose accelerated synthesis is provoked by: (i) carbon or nitrogen starvation or (ii) carbon or phosphate starvation (figure 3). Amongst the Sti proteins of E. coli two sub groups can be identified on the basis of whether or not their synthesis is influenced by the intracellular concentration of cyclic AMP. Starvation of E. coli causes an increase in concentration of this signal molecule which, among other actions, stimulates transcription of a sub-set of sti genes responsible for specifying Cst proteins. This leaves other sti genes (encoding Pex proteins) whose transcription is not under the control of CAMP-regulated promoters. The Pex proteins are at the heart of

Multiple starvation specific proteins

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prZ:ins

cs Carbon starvation stimulon

1

CNs proteins

proteins

gs proteins

Ns proteins NPs proteins

I

I

Nitrogen starvation stimulon

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Ps proteins

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Phosphorous starvation stimulon

Figure 3 Grouping of starvation-induced (Sti) proteins into different overlapping starvation stimulons in the marine bacterium Vibrio sp. S14. (From Nystrijm et al. [20], reproduced courtesy of Elsevier Science Publishers.)

the core starvation response which is elicited irrespective of the actual nutrient deficiency that provokes it, and identifiable amongst them are the protein products of a KatF regulon [ 18, 191. First recognized as a regulatory gene implicated in the production of certain specific proteins (including a catalase) during the stationary phase of batch culture growth, k&F was found to encode a protein whose structure is very similar to the normal sigma factor (070) of E. coli. It was therefore presumed that KatF is a starvation-specific sigma factor so that it might be more logical to designate kutF as rpoS. Under the control of KatF (RpoS) are genes that specify a hydroperoxidase HPII, a repair exonuclease III, a scavenging acid phosphatase and enzymes involved in the synthesis of trehalose whose accumulation in E. coli can be induced in response either to starvation or to hyperosmotic shock. Elements of the starvation shock response are identifiable even in those bacteria alluded to earlier that, as ultramicro cells, can survive prolonged periods of nutrient deprivation. Thus the marine bacterium Vibrio sp. 14 when subjected to a starvation shock responds with a transient expression of the (recA dependent) stringent response, sequential synthesis of Sti proteins, increased resistance to lysis, heat shock and ultra violet radiation, reduction in metabolic activity, cessation of DNA synthesis and secretion of scavenging exoproteases [20]. Conclusion It is intriguing that the mechanisms for countering stress were evidently developed early in the history of bacteria, and were manifestly so successful that they became universally distributed among prokaryotes and were conserved during the evolution

of other life forms. The mechanisms are elegant in their simplicity (sensors signalling via activation of transcriptional regulators) and effective in their outcome (a core global response with protective and repair elements sustaining viability while more specific steps are taken to combat the actual stress that is being encountered). The manner in which these elements are combined, the patterns of control and of ‘crosstalk’ between regulons, and the fields of overlap between stimulons, provide fertile ground for experimentation directed at improved understanding of the networking and orchestration of cellular events. But there are other good reasons why we should seek to discover more of the molecular bases of bacterial stress responses: for example, because of the part they may play in helping to counter the natural antibacterial machinery of an infected host organism, or because we might be able to exploit their properties for biotechnological purposes. Thus a prime function of white blood cells (macrophages) is to ingest and destroy invasive bacteria. The interior of such a macrophage should therefore be a most uncongenial environment for any ingested bacterium, which would encounter therein an acidic pH, oxidative free radicals, toxic lysosomal peptides, etc. Yet some disease-causing bacteria can so successfully combat all of these stresses that they can take up ‘permanent’ residence in these leucocytes. It has been noted that when S. typhimurium infects macrophages the synthesis of oter 30 bacterial proteins is selectively induced. From what we now know of stress responses, it comes as no surprise to us to learn that amongst these macrophage-induced proteins (MIPS) are DnaK and GroEL which serve as immunodominant antigens. The synthesis of MIPS is there5

fore not only of significance in determining whether an invading bacterium is destroyed or will survive within a macrophage,but may also prove in some instancesto be one factor in the triggering of host autoimmunity. Finally, somepractical use may be made of the regulatory elementsof the stressresponse genes i’n constructing genetically engineered bacteria. For example, by putting recombinant genes under the control of pex-type promotersit might be possible to delay expression of these genes until batch cultures have entered their stationary phaseswhen the bacterial population is at its most dense and supplied sourcesof carbon and energy (or specific biosynthetic precursors)will not be preferentially diverted to the needs of growth and reproduction. Or again, benefit could accrue from the use of such starvation-responsive elementsto control expression of catabolic genes introduced into indigenous, naturally slow growing (autochtho-

nous) bacteria which, in some instances better than less robust if more spectacular species,could effect the progressivebioremediation of polluted environments.

References [l] Neidhart, F. C. In Escherichia coli and Salmonella typhimurium Cellular and

MolecularBiology (Ed.F. C. Neidhardtet ~2.).p. 1313et seq., AmericanSocietyfor Microbiology, Washington, DC, 1987. [2] Roszak, D. B. and Colwell, R. R. Microbial. Rev., 51, 365, 1987. [3] Koshland, D. E., Jr. ‘Bacterial Chemotaxis as a Model Behavioural System’, Raven Press, New York, 1980. [4] Armitage, J. P. A. Rev. Physiol., 54, 683, 1992. [5] Stock, J. B., Surette, M. G., McCleary, W. R. and Stock, A. M. J. biol. Chem., 267, 19753,1992. [6] Ritossa, F. Experientiu, 18,571, 1962. [7] Ang, D., Liberek, K., Skowyra, D., Zylicz, M. and Georgeopolous, C. J. biol. Chem., 266,24233,1991.

[8] Nilsson, B. and Anderson, S. A. Rev. Microbial., 45,607, 1991. [9] Craig, E. A. and Gross, C. A. Trends Biochem. Sci., 16, 135, 1991. [lo] Murray, P. J. and Young, R. A. J. Bacr., 174,4193,1992. [ 1 l] Garbe, T. R. Experientia, 48,635, 1992. [12] Yang, X-D. and Feige, U. Experientia, 48, 650,1992. [13] Farr, S. B. and Kogoma, T. Microbial. Rev., 55,561, 1991. [14] Demple, B. and Amabile-Cuevas, C. F. Cell, 67,837, 1991. [15] Storz, G., Tartaglia, L. A. and Ames, B. N. Science, 248,189,1990. [16] Wu, J. and Weiss, B. J. Butt., 173, 2864, 1991. [17] Matin, A., Auger, E. A., Blum, R. H. and Schultz, J. E. A. Rev. Microbial., 43, 293, 1989. [18] Siegele, D. A. and Kotter, R. J. Back, 174, 345, 1992. [19] McCann, M. P., Kidwell, J. P. and Matin, A. J. Bucr., 173,4188, 1991. [20] Nystrijm, T., Albertson, N. H., Fl%rdh, K.1 and Kjellenberg, S. FEMS Microbial. Ecoi., 74, 129, 1990.