cycles of famine and feast: the starvation and outgrowth

Cycles of famine and feast: the starvation and outgrowth strategies of a marine Vibrio

SUJATHA SRINIVASAN and STAFFAN KJELLEBERG* School of Microbiology and Immunology, University of New South Wales, Sydney 2052, Australia

*Corresponding attthor (Fax, 61-2-9385 1779; Emaif, [email protected]).

Studies of starvation survival in non-differentiating bacteria have largely focused on physiological changes and regulatory aspects of a few master regulators such as the signal molecule ppGpp and the stationary phase alternative sigma factor, sigma S. Recent findings have implicated a series of novel key events for the entry as well as exit from starvation. The importance of alternative sigma factors other than sigma S is emerging. In addition, low moIecular weight extracellular signals have been demonstrated to be essential for the induction and mediation of several adaptive responses. The importance of rnRNA modification and stability for starvation survival as well as outgrowth is receiving renewed interest. In this paper, we present the results obtained from studies of starvation survival and recovery of Vibrio sp. strain SI4.

1. Introduction

Bacteria are constantly challenged by conditions of nutrient limitation and starvation in natur~tI environments (Moriarty and BelI 1993; Morita 1993). As bacteria cannot escape the environment, they undergo changes in their phenotypic and genetic repertoire in order to adapt to the specific condition. The bacterial response to starvation is not simply an arrest of all metabolic activity. It can be argued that the life cycle of bacteria broadly consists of two major phases, the growth phase and the starvation or stationary phase. The transition between these two phases involves dramatic changes in gene expression, physiology and morphology. Some bacteria escape harsh conditions by differentiating into starvation- and stress-resistant forms such as spores and fruiting bodies (Grossman 1995; Kaplan and Plamann 1996). On the other hand, studies conducted over approximately the last 30 years have demonstrated that many non-differentiating bacteria have the ability to withstand starvation by altering their physiological response.

In the marine environment, sporadic growth would appear to be the norm rather than the exception. Bacteria lead a 'feast or famine' existence where intermittent short periods of growth are complemented by extended

periods of starvation (Kjelleberg er al 1993a). Members of the family Vibrionaceae are widespread in the marine environment and represent a major part of the culturable fraction. Additionally, they have the abiIity to proliferate in areas of high substrate availability and can persist as free-living cells during conditions of nutrient limitation, thus making them highly suitable for starvation studies (0stling et al 1993).

This review will focus on the starvation-survivaI and recovery strategies of Vibrio sp. strain S14. Given the multidisciplinary nature of the stress workshop held at Varanasi, this presentation will attempt to provide an overview as well as an insight into the physiological and molecular mechanisms that orchestrate the adaptive response in Vibrio sp. S14. Particular emphasis will be given to the role of exometabolites as signal molecules in the starvation adaptation programme.

2. The organism

Vibrio sp. S14 is a marine, heterotrophie, Gram~negative, motile and curved rod (Humphrey et at 1983). it is facultatively anaerobic with a temperature optimum of 26~ The 16S rRNA sequencing and biochemical tests have shown that this bacterium has a high degree of

Keywords. Marine Vibrio; carbon starvation; outgrowth; recovery; stringent response; extracellular signals

J. Biosci., 23, No. 4, October I998, pp 501-511. �9 Indian Academy of Sciences 501

502 Sujatha Srinivasan and Staffan Kjelleberg

similarity to Photobacterium angustum (S Zabkar and S Kjelleberg, personal communication). Many studies have demonstrated that Vibrio sp. S14 is capable of systematic cellular reorganization on encountering nutrient limitation and can respond immediately to substrate availability. It has the ability to survive at least 6-5 years in a carbon- and energy-depleted environment (13stling 1996).

3. Adaptation to nutrient limitation

3.1 Changes in the morphology and physiology

3.1a UltramicroceH formation: Pronounced morphological changes occur in response to starvation. A dep- rivation in carbon and energy sources results in the formation of ultramicrocells. This ultramicrocell formation is a consequence of reductive cell division, wherein cells complete ongoing rounds of replication and undergo cell division without biomass increase, generating daughter cells with at least one chromosome. There is a school of thought that reductive division allows for the increase in the number of progenies thus augmenting the possibility of the survival of at least one cell. In theory, only one cell per species is required for the preservation of that species (Morita 1993). It has been argued that the ultramicrobacteria may have a competitive advantage in the marine environment as they may not be under the same grazing pressure as larger cells, thus being protected against predation by protozoa (Morita 1985).

Recent electron microscopy studies of Vibrio sp. S14 have revealed that after 24 h of starvation for a carbon and energy source, there is a heterogeneous population of rod and coccoid cells with or without flagella. After 3 days of starvation, majority of the cells were non-motile, without flagella and surrounded by electron dense material. However, after 16 days of starvation, no electron dense material was detected (Stretton et al 1997). This indicates that the initial response to starvation is not homogeneous within the population. On the other hand, under conditions of long-term starvation, the response of the entire population is uniform.

3.1b Phases of starvation: The morphological changes that occur as a result of cells encountering starvation do not exhibit any defined stages. Based on the sequential course of physiological events taking place in response to nutrient depletion, however, three phases in the adaptation pattern have been identified; the stringent control phase (0-30 min), the intermediate reorganizational phase (30min-6h) and the long-term starvation phase. For further cletaiIs on the specific changes in the different phases, the reader is referred to reviews by Kjetleberg et al (1993b) and ()stling et al (1993).

3.1c Carbon starvation--the determinant for successful adaptation to starvation: A systematic study of the

responses of Vibrio sp. $14 to multiple-nutrient (cells starved simultaneously for carbon, nitrogen and phosphorus), carbon, nitrogen or phosphorus starvation has been conducted. The adaptation patterns in response to multiple-nutrient and carbon starvation are similar while significant differences have been observed in adaptation patterns to nitrogen and phosphorus starvation.

Starvation- and stress-resistant ultramicrocell formation occurs only in response to carbon and multiple-nutrient starvation (NystrOm it al I992; Holmquist and Kjelleberg I993). Nitrogen limitation leads to the formation of thin filaments while phosphorus starvation results in .swollen elongated cells. Multiple-nutrient or carbon-starved cells remained I00% viable during one week of starvation as determined by direct plate counts. On the other hand, nitrogen- and phosphorus-starved cells exhibited a significant loss in plateability after just 2-3 days. Yet, prolonged incubation of the plates revealed the formation of microcolonies in approximately the same numbers as observed at the onset of starvation (Holmquist and Kjelle- berg 1993). Carbon and multiple-nutrient starvation lead to a shut-down in the synthesis of RNA and proteins while nitrogen and phosphorus starvation do not exhibit this stringent control (0stling et al 1993). Data obtained from two-dimensional polyacrylamide gel electrophoretic (2D-PAGE) analyses of proteins in response to the different starvation conditions indicated that there was not much overlap between proteins expressed as a result of carbon and energy starvation and starvation for nitrogen or phosphorus (0stling 1996). The above resuIts suggest that carbon starvation is the determinant for successful formation of starvation- and stress-resistant ce?Is in Vibrio sp. SI4.

3.1d Expression of starvation-induced proteins: The large number of proteins that respond to a given environmental stimulus in Vibrio sp. S14 have been studied by 2D-PAGE. This method has generated a wealth of information whereby the phenotypic response to different environmental conditions has been mapped and analysed. The approach has been particularly useful in our studies of the starvation adaptation patterns of Vibrio sp. S14 as it permitted a global investigation of an elaborate response.

In Vib~qo sp. $14, the starvation-induced proteins are induced in a time dependent fashion in response to multiple-nutrient starvation. These proteins can be grouped into different classes according to their time of appearance during starvation. The proteins are synthesized in a burst in the initial phase. Inhibition of protein synthesis by the addition of chloramphenicoI in this phase can have a lethal effect on the viability of the cells (Nystr0m et al 1990). This indicates that expression of starvation-induced proteins in the initial phase is important for starvation-survivaL The process of sequen-

Starvation and outgrowth cycle in marine Vibrio 503

tial expression of proteins is similar to programmed expression of unique genes in differentiating bacteria.

Initial studies suggested that there is a sequentiaI expression of about 60 proteins in response to multiple- nutrient starvation. In the first phase, at least 38 proteins were increased followed by 15 in the second and 8 in the third phase. Some were transiently induced while others were permanent during the 100 h period (Nystr6m et al 1990).

Recent studies have demonstrated that the adaptation to carbon starvation involves a much larger phenotypic expression. Improved 2D-PAGE procedures have permitted detection of more than i700 proteins (13stling 1996). The relative rate of protein synthesis was significantly increased for as many as 157 proteins and decreased for 144 proteins after I h into carbon starvation (Ostling et al I996). Standard gels have been established in order to facilitate comparison between different conditions (Ostling 1996).

3.2 Regulatory features of carbon starvation adaptation

Contemporary studies of the starvation response have focused on understanding its regulation. In tight of the complex shifts in protein synthesis, an intricate regulatory network is more than likely to exist. Several lines of evidence indicate that regulation of carbon starvation involves more than one element. Figure l outlines the various regulatory aspects of the carbon starvation response in Vibrio sp. S14+

3.2a The rote of stringent control: The stringent response, as studied in Escherichia coli (Cashel et af 1996), is induced as a result of amino acid starvation when the amount of amino-acylated tRNA in the cell is not sufficient for protein synthesis. The response involves the accumuIation of guanosine 3'-diphosphate 5'-diphosphate (ppGpp) which is synthesized by the enzyme RelA (ppGpp synthetase). Subsequently, ppGpp is degraded by the enzyme SpoT (ppGpp 3'pyrophos- phohydrolase). Increased intracellular concentrations of ppGpp immediately result in decreased gene expression and hence translation. It has been argued that this control mechanism prevents the cell from synthesizing aberrant peptides when the availability of amino acids is restricted. Eventually, the stringent response up-regulates the genes involved in biosynthesis of amino acids, allowing the pool of amino acids to be restored. RelA activity decreases and stringent control is relieved. However, it has been shown that ppGpp concentration increases not only in response to amino acid starvation but also as a result of glucose starvation (Chaloner-Larson and Yamazaki 1978). It is believed that glucose starvation brings about a transient amino acid starvation which then triggers

RelA to catalyse ppGpp formation. SpoT can reverse its activity specifically in response to glucose starvation and catalyse the formation of ppGpp (ppGpp synthetase II activity) (Xiao et al 1991).

The stringent response involving RelA and Spo t is perhaps one of the best studied features of the starvation response in Vibrio sp. S14 (Fl~irdh et al 1994; Ostling et at 1995, 1996). Accumulation of ppGpp can be observed during amino acid and energy source starvation (Fl~irdh et al I994). Both relA and spoT mutations have been obtained (Flardh et al 1994; Ostling et al 1995). In response to carbon starvation, the relA mutant and the wild type display the same degree of starvation survivaI and stress resistance (Fl~irdh et al 1994). On the other hand, the spoT mutant is impaired in its ability to survive _during carbon starvation and is sensitive to stress conditions like UV irradiation and visible light (Ostling et al 1995). Measurements of the ppGpp levels in Vibrio sp. S14 refA mutant have indicated that SpoT only makes a small contribution to the accumulation of ppGpp observed in response to glucose starvation.

Comparison of 2D-PAGE data obtained from relA, spoT and wild type strains revealed that there was little difference in the translational products obtained in the exponential phase. On the other hand, large changes in protein expression were observed in response to carbon starvation and most differences resided in the carbon starvation-induced ([C.IND]) and carbon starvation- repressed proteins ([C.REP]) (13stling et al 1996).

Early studies have shown that Vibrio sp. S14 induces a high affinity glucose uptake system in response to starvation (Albertson et al 1990a). On comparison of the glucose uptake systems in relA, spoT and wild type strains, it was found that the spoT strain displayed a 20% decrease in the Vm~ of the uptake system. The relA strain did not exhibit any major differences when compared with the wild type (Ostling et al 1996).

These findings suggest that SpoT may play an important rote as a carbon starvation-specific regulator in a ppGpp- independent manner (Ostling et al 1995, 1996). RelA- mediated ppGpp synthesis appears to be important in optimal performance of the cells during conditions of nutrient limitation particularly in the earlier stages of adaptation (Fl~irdh et al 1994). It has therefore been hypothesized that relA-mediated ppGpp accumulation and an additional, essential signal mediated by spoT are required for the complete phenotypic expression in re- sponseto carbon starvation (0stling 1996). Further studies will address the identity and the role of the carbon starvation genes that are uniquely induced by SpoT as they are likely to be the core of the carbon starvation genes essential for survival.

3.2b Regulation by sigma factors: RNA polymerase hotoenzyme of E. coli consists of the core enzyme with the subunit composition a:~fl' and a o subunit which


LOW g l u c o s e ~

|

% ,r �9 ~ s" I

~,-.~,- :,~ I I I ! I I i

Fz;1.-| exponen t i a l s__ stringent J

growth response

%

( poly~A) ~ HIGH

_I_ .: : - ....

" - , I i i i ! ! ! ! !oio0 & o

" " " - - " - - ~

�9 ~ of stress resistance

S t i s (>157)

Figure 1. Model for the carbon starvation and outgrowth cycle in Vibrio sp. S14, The figure outlines specific regulators, protein responders and pathways of regulation studied. The shaded rod shaped ceil represents a logarithmic cell before entry into carbon starvation and the shaded coecoid cell, an ultramicroce[1 primed for the outgrowth response. Unshaded and shaded triangles and circles on the bacterial cells indicate low and high affinity for glucose and amino acids, respectively. The shaded ovals are regulators namely RelA, SpoT, RpoE, RpoS and Csf. The rectangles depict signal molecules. Large circles are starvation (Stis) and immediate upshift (lups) protein responders. Overlap between the Sti circles indicates the protein responders induced by both RelA and SpoT. A carbon starvation factor (Csf) has been hypothesized to be an intermediate between SpoT and Stis. 157 Stis and 18 Iups have been mapped and analysed by 2D-PAGE. mRNA (--~) modification and stability has been proposed to be modulated by RNase E (unshaded oval), mRNA binding proteins (small hatched oval) and polyadenylation (polyA taiI). For details of the regulation of" the carbon starvation and outgrowth response in Vibrio sp. SI4, please refer to the text.

directs the core enzyme to initiate transcription at specific promoter sites on DNA. There are different types of cr factors of which the alternative a factor, a 3s (or os), is a key factor in the starvation and stress response occurring during the transition from exponentiaI growth to stationary phase. For details the reader is referred to reviews by Hengge-Aronis (1993, 1996a,b), The rpoS gene encodes the sigma factor o s which has been hypothesized to be a central regulator in a complex regulatory network that governs the expression of many stationary phase or starvation-induced genes (Lange and

Hengge-Aronis 1991, 1994). Interestingly, o s has also been implicated as a global regulator for the osmotic control of gene expression in exponentially growing cells. As the role played by o s is not confined to the stationary phase, it has been debated that o s may be viewed as a sigma factor associated with general stress conditions (Hengge-Aronis 1996a).

The amount of o s in the cell has been proposed to be regulated at the levels of transcription, translation and protein stability (Lange and Hengge-Aronis t994). The o s protein has been found to have a short half-life


in the exponential phase primarily due to the presence of a specific protease, the ClpXP protease (Schweder et at 1996). Many other factors have been proposed to control the level of a s in the cell. It has been demonstrated that ppGpp is a positive regulator of o s synthesis at the tevel of rpeS transcription (Gentry et at 1993) and that RNA polymerase hr~s a binding site for ppGpp (Reddy et al 1995). It has been suggested that the historic-like protein H-NS may modulate the level of a s in the cell by acting as a global regulator of gone expression during the exponential phase (Barth et al 1995). cAMP-CRP complex may negatively control the transcription rate (Lange and Hengge-Aronis 1994). In addition, UDP- glucose (BOhringer et at 1995) and homoserine lactone (t-ISL) (Huisman and Kolter i994) have been proposed to function as intracellular signals which play a role in the expression of as and as-dependent genes.

In addition to o s, several other ~r factors function as crucial regulators in response to various stress conditions. Some examples are the as2 which has a role in the heat shock response and in increased cellular resistance to starvation in E. coli (Gross I996; Yura et al t993) and o ~4 which is important during nitrogen starvation in E. coU and which has also been shown to regulate a gone concerned with carbon starvation survival in Pseudomonas putida (Kim et al 1995). Recent investigations have revealed that the alternative sigma factor a E is also involved in regulation of the heat shock response, particularly at extreme temperatures (Hiratsu et al 1995; Rouvi~re et at [995). Apart from being activated by heat or ethanol, er e is induced by disruption in protein ~blding in the periplasm (Mecsas et al 1993; Missiakas et al 1996). Moreover, in the deep-sea bacterium Photo- bacterium sp. strain SS9, the rpoE gone product was shown to influence outer membrane protein synthesis and also played a role in modulating growth at high pressures and cold temperatures (Chi and Bartlett 1995). Strikingly, all o ~ hemologues identified to date transcriptionally regulate genes encoding proteins with extracytoplasmic functions and responding to extracytoplasmic stimuli (Lonetto et al 1994). Interestingly, a recent study investigating the cellular roIe of o ~ in E. coli demonstrated that o -B is essential for growth at least at temperatures of 18~ or more (Pefias et al 1997).

Many stimulons induced by stress conditions have also been induced by carbon starvation (Nystr~Sm et al 1990; Matin 1991; Spector and Foster 1993; Givskov et at 1994). This indicates that the stress response regulation to any specific condition is achieved by more that one alternative cr factor. In Vibrio sp. S14, studies addressing the role of key a factors in the carbon starvation response have been initiated. The Vib~io Sl4tpoE (o I~) gene has been cloned and sequenced. Its cellular role is currently being studied by mutational analyses (E Hild and S KjeIIeberg. unpublished), tt is pertinent to note that in

Vib~4o S14, there are significant overlaps in the outer membrane proteins (OMPs) induced by starvation, ethanol and heat shock (Nystr0m et al I988). Given that more than one alternative ~ factor may be involved in regulating a stress condition and that there are overlaps in the OMPs induced by starvation, ethanot and heat shock, we hypothesize that the carbon starvation response in gibtio sp. S14 may elicit the induction of both o ~ and o s.

3.2c The role of extracethdar signal molecules: So far, much emphasis has been placed on understanding the starvation response in bacteria in terms of the physiological and molecular alterations. However, the means by which a cell senses the environmental change and transduces this change to an internal signal in a language that can be interpreted biochemically remains largely unknown. Fortunately, there is a current interest in identifying extraceltular molecules and determining their role in adaptive responses.

(i) The case ht differentiating bacteria: The role of exometabolites facilitating chemical communication in differentiating bacteria is well established. In actinomycetes, y-butyrolactones function as autoregulators which at nanomolar concentrations control morphoIogical differentiation (aerial mycelium formation) and secondary metabolite production (Bibb 1996; Horinouchi and Beppu 1992, 1994). In Myxococcus xanthus, multicellular fruiting body development requires the transmission of inter- ceiIular signals of which two namely factors A and C have been analysed in great detail. The C-signal is a cell-associated protein requiring cell-cell contact for signal transmission (Kaiser and Kroos 1993) unlike the other signal molecules being considered in this paper which are small organic molecules with the ability to freely diffuse into the surrounding medium. The A signal, on the other hand, functions as a celt density signal during starvation that permits development to proceed once a threshold concentration has been achieved. The A signal comprises of a specific set of amino acids that functions at a concentration greater than 10 I-tM (Kaplan and Pla- mann 1996). In addition, the E-signal, which consists of one or more branched-chain fatty acids, needs to be transmitted betweeit cells to complete development in M. xanthus (Downard and Toal 1995). Although the E-signal requires cell-cell contact, it is pertinent to men- tion that the concept of fatty acids being involved in cell-cell communication is rather novel. Sporulation and acquisition of competence for DNA transformation in Bacillus subtitis is dependent on oIigopeptides that accumulate in the medium. Once a critical concentration is reached, the ceils enter stationary phase and subsequently differentiate into starvation-induced spores (Grossman 1995; Magnuson et at t994).

505 St,jatha Srinivasan and Sraffan Kjelteberg

(ii) The case in non differentiating bacteria: Several examples of small molecules mediating gene expression in non-differentiating bacteria have emerged. Acylated homoserine lactones (AHLs) occur in many Gram- negative bacteria and control a variety of ceil density dependent factors; the best characterized being bacterial bioluminescence (Fuqua et aI 1996; Fuqua et al 1994; Gray 1997; Swift et al 1994, 1996). Two proteins, LuxR and LuxI play central roles in the regulation of the lux operon in Vibrio fischeri. LuxI functions as the signal generator from S-adenosyl methionine and acyl-ACP (acyl carrier protein) from the fatty acid biosynthetic pathway (Mor6 et al 1996). The signal molecule, in this case N-(3-oxohexanoyt) homoserine lactone, binds to the LuxR protein (Schaefer et al 1996) which functions as a transcriptional activator of luxl and lux structural genes. Autoinducer-bound LuxR down-regulates expression of the luxR gene.

As the phenomenon of quorum sensing is typically associated with the onset of the stationary phase, the possibility of rpoS expression being linked to ceil-cell signalling was examined. Recent research has demonstrated that the LuxR-like transcriptional activator RhlR may be involved in regulating rpoS ~n Pseudamonas aeruginosa (Latifi et al I996). Furthermore, rhlR expression is controlled by LasR (a LuxR homologue) and expression of rpoS is abolished in a lasR mutant in P. aeruginosa. Thus, it appears that quorum-sensing in P. aerugbzosa involves a cascade that engages both RhlR and LasR as transcriptional activators with the stationary phase sigma factor forming part of the equation (Latifi et al 1996). Moreover, HSL has been proposed to be a key signal for inducing the transcription of rpoS in E. coll. It was suggested that ppGpp controls the expression of o s via HSL which acts as an intermediate signal (Huisman and Kolter I994). The induction of stationary phase physiology by the addition of putative signals present in the supernatant was also observed in Rhizobium legu- minosarum (Gray et al 1996).

There have been several investigations to determine the role and identity of novel extraeellular molecules in E. coll. The exometabolite 1,5-anhydroglucitol has been proposed to be a metabolic signal of nutritional conditions (Shiga et af 1993). As yet unidentified exometabotites, produced under different stress conditions in E. coil, provided 'cross-protection' against other stresses such as heat shock and oxidative stress (Nikolaev I997). Sta- tionary phase supernatant extracts of E. coli inhibited SdiA-mediated expression of ftsQAZ, an operon whose genes encode functions required for celI division. SdiA is a member of the LuxR family of transcriptional activators (Garcia-Lara et al 1996). Cyclic dipeptides (diketopiperazines) have been isolated from stationary phase supernatants in E. coli and the effect of these low molecular weight secondary metabolites on the expression

of the E. coli stationary phase genes rpoS, bolA, t ic and f tsQA was examined. The cyclic dipeptides down-regulated the activity of bolA and t ic (RpoS-regulated genes) while they had no effect on rpoS and f tsQA (R de Nys, K Yamamoto, M Givskov, N Kumar, R Read, R Utsumi and S Kjelleberg, unpublished). Cyclic dipeptides have also been found in the cell-free supernatants of P. aeruginosa, Proteus mirabilis, Citrobacter freundii, Pseudo- monas fluorescens, Pseudomonas alcaligenes and Enterobacter agglomerans (S R Chhabra, M T G Holden, P Stead, N I Bainton, P J Hill, G P C Salmond, G S A B Stewart, B Bycroft and P Williams, unpublished). Although the role played by these molecules is yet to be determined, their widespread occurrence may indicate that they are a new class of signalling molecules.

Other cases where bacteria employ low molecular mass diffusible signals in order to influence gene expression have been reported. The Gram-negative plant pathogen Ratstonia (Pseudomonas) solanacearum produces a vola- tile extracellular factor namely, 3-hydroxypalmitie acid methyl ester (3-OH FAME) (Flavier et al 1997a). This compound was found to autoregu]ate the expression of virulence factors and is an interesting example of a fatty acid derivative that can mediate long-distance intercelIular communication (Clough et al i997; Ftavier et al 1997a). Moreover, R. solanacearum produces N-hexanoyl and N-octanoyl homoserine lactones and these AHLs are under the control of phcA (a LysR type transcriptional regulator of virulence determinants). PhcA is regulated by 3-OH PAME and hence the AHLs are part of an hierarchical signal transduction pathway. While the role of AHLs is yet to be determined, it has been demonstrated that eliminating AHL production did not affect the virulence of the plant pathogen. The expression of aidA, a gene linked to sotR and soll (tuxR and luxl homologous) was found to be dependent on the AHLs. However aidA lacks homology to other genes in the databases and hence, this gone did not provide any cIue to the role played by the AHLs (Flavier et al 1997b). Another example of extraeeitular signals in different bacterial species is that of Xanthomonas campestris in which a low molecular weight diffusible factor, unrelated to AHLs, regulates the expression of extracelIular enzymes and polysaccharide virulence determinants (Barber et at 1997). In addition, extracellular factors at concentrations of ng ml -~ have also been reported to allow for resuscitation from dormancy in Micrococcus luteus (Kaprelyants and Kell 1996).

(iii) The case in Vibrio sp. $14: The role of exometabolites as putative signal molecules in the carbon starvation response was investigated in Vibrio sp. S14. Two-dimensional polyacrylamide gel electrophoretic (PAGE) analysis revealed that addition of the stationary phase supernatant extract (SSE) of Vibrio sp. S14 to exponential


ceils led to the up-regulation of a significant number of carbon starvation-induced ([C.IND]) proteins. A remark- able 56% of proteins normally repressed upon carbon starvation were found to be down-regulated, indicating that the exometabolites wei-e affecting significant

sub-populations of the carbon starvation stimulon (Srini- vasan et al 1998).

Furthermore, the addition of halogenated furanones, specific antagonists of AHLs (Givskov et al 1996; Gram et al 1996), resulted in the repression of 30% [C.IND] proteins and induction of I7% carbon starvation-repressed ([C.REP]) proteins when the furanone was included in the starvation regimen. Moreover, 78% of proteins that were inhibited by the furanone were carbon starvation- specific. With respect to the up- and down-regulation of protein expression, the effect of the furanone was opposite of that caused by SSE, indicating that both the furanone and putative signalling molecule(s) were acting on the same regulatory pathway. It was determined that 12 proteins were common to the [C.1ND] proteins up- regulated by SSE and the proteins down-regulated by the furanone. Of the 12, 11 proteins (92%) were found to be carbon starvation-specific. Importantly, there was no significant overlap between proteins affected by the furanone and proteins of the nitrogen and phosphorus starvation stimuIons. This is congruent to previous studies in our laboratory demonstrating that only carbon starvation allows the cell to elicit the differentiation-like adaptation programme. The addition of the furanone to exponential cells, on the other hand, resulted in the down-regulation of only 3 out of the 637 proteins compared, again suggesting that the furanone was specifically affecting the proteins of the carbon starvation stimulon.

Addition of the furanone to carbon starvation regimens reduced viability (assessed by colony forming units) and abolished the development of stress resistance against UV irradiation and H20~ " exposure, the hallmarks of the starvation response of Vibrio sp, S14. However, when the furanone was added to exponentiaI cells at the same concentration, there was no effect on the growth rate, thereby suggesting that the furanone was interfering with a system essential tbr st,arvation adaptation. In another experiment, to further explore whether components in the SSE and the furanone were acting on the same regulatory pathway, the culmrabflity of gibrio sp. SI4 during carbon starvation was tested in the presence of both SSE and the furanone. Indeed, it was found that the SSE was able to provide protection against the loss in viability caused by the furanone (Srinivasan et al 1998). Hence, it can be concluded fi'om the evidence presented by the above observations that extraceIIular signals mediate the synthesis of carbon starvation adaptive protein responders and the development of starvation and stress-resistant ultramicrocells in Vibrio sp. S14,

The identity of the proposed signal moIecule(s) is

presently being considered. In spite of the model AHL phenotypes, namely swarming in Serratia liquefaciens and bioIuminescence in Vibrio harveyi, being induced by the addition of supernatants from carbon starved celIs, no AHLs were detected in the SSE (Srinivasan et al I998). However, this is not surprising as other low molecular weight compounds have been found to interfere with AHL-reguIated phenotypes. Cyclic dipeptides have been shown to interfere with the AHL-med~ated phenotypes of bioIuminescenee in Vibrio f ischeri and swarming in S. tiquefaciens (unpublished results). Furthermore, the SSE of Vibrio vttlnificus, which does not contain AI-ILs, had the ability 'to protect' Vibrio sp. $14 cells from loss in viability caused by the furanone molecule during carbon starvation, indicating that similar exometabolites capable of 'cross-talk' exist in this organism (unpublished data). This phenomenon of 'cross-talk' between several types of extracellular molecules certainIy has far reaching ramifications when adaptive responses in the natural environment are considered.

4, The outgrowth response

The abiIity of the starved cell to resume growth when nutrients become available is critical in determining its competitive ability in nutrient-limited environments and hence the ultimate survival of the species. A recent study of the morphoIogical and physiological changes occurring in response to nutrient addition (complex medium) after long-term starvation (16-18 days) in Vibrio sp. S14, showed that the optical density of the cuIture increased after a 1.5 h lag. The viable counts (measured as colony forming units) increased after a 2.5 h lag from 3% to 30% of the total count over a period of 20 rain. Electron microscopy data indicated the presence of 2 populations of which 75% were large, rod shaped and intraeellularly dense while 25% were microcells, indicating that the response was not homogeneous. Phase contrast microscopy data revealed that 20% of the total celIs were motile 2 h after nutrient addition while 41% were motile after 5 h. Further analysis by electron microscopy to determine tile presence of flagella demonstrated that only red eelIs were flagellated while the microcells ren~ained non-f[ageItated (Stretton et al 1997).

Vibrio sp. 814 has the capacity to instantaneously respond to carbon (glucose) addition by increasing the rate of RNA and protein synthesis after Iong-term starvation (Albertson et al i990b; Marouga and Kjelleberg 1996). This conservation of a functional protein synthe- sising machinery is apparently independent of the duration of starvation although the subsequent lag phase (or maturation phase) is dependent on the length of the starvation period. Analysis of proteins by 2D-PAGE indicated that starvation-induced proteins were repressed

508 Sujatha Srinivasan and Staffan KjeUeberg

after nutritional upshift of cells starved for 24 h. Twenty proteins were induced at various intervals upon upshift of which 10 were maturation phase-specific proteins (Albertson et at 1990b).

Recent studies of the outgrowth response in Vibrio sp. S14 have shown that there is an immediate upshift phase of 3 min after cells have been starved for a glucose source for 48 h. The response is manifested with the induction of I8 proteins designated as immediate upshift (!up) proteins. Some of the Iup proteins were transiently induced while others were found to be induced for at least 60 rain after the onset of recovery. Experiments based on rifampicin addition prior to glucose supple- mentation demonstrated that some of the i~p transcripts were long-lived (Marouga and Kjelleberg 1996).

The up-regulation of protein synthesis observed in response to nutritional upshift has been shown to be independent of de novo RNA synthesis (Albertson et al 1990b; Marouga and Kjelleberg 1996). It has been argued that the presence of stable and active transcripts during carbon starvation (Albertson et al 1990c) and general s'tabilization of mRNAs by decreased endonucleolytic activity and mRNA binding proteins (R Marouga, D Georgellis, B Sohlberg, A van Gabain and S Kjelleberg, in preparation) may allow for stable but silent transcripts to be post-transcriptionally activated upon nutritional upshift. Moreover, mRNA poIyadenylation has been suggested to play a possibte role in suppression of specific proteins during recovery by facilitating mRNA degrada- tion (K Takayama, M Oramas and S Kjelleberg, personal communication). The observation that ribosomes exist in large excess of the apparent demand for translation in carbon starved cells may additionally explain the efficient resumption of protein expression upon recovery (Flardh et al 1992). Interestingly, the mean mRNA half-life immediately decreased when 24h starved cells were provided with glucose minimal medium and within 5 h of re-growth, the value was similar to that in growing cells (Albertson et al 1990c).

Study of some of the regulatory aspects of the recovery response has shown that the stringent response is elicited after carbon source upshift. This stringent controI is dependent on the relA gene as the relA- strain exhibited a prolonged lag phase. Addition of glucose to Vibrio sp. S I4 after 48 h of carbon and energy starvation causes amino acid limitation and induces the stringent response after 4 rain. The amino acid limitation appears to be caused by the rapid increase in the rate of translation upon upshift which consumes the cellular amino acid supplies. It has also been argued that there is a repression of amino acid biosynthetic enzymes during carbon starvation and these are based on observations that although protein synthesis accelerates within 2 min on addition of glucose, incorporation of sulphur containing amino acids does not occur until after 2 h (Flardh and Kjelleberg

1994). A model of the outgrowth response in Vibrio sp. S14 is presented in figure 1.

5. Concluding remarks

Bacteria have evolved strategies to survive the stressful condition of nutrient limitation in natural environments. Moreover, they have developed suitable mechanisms to overcome starvation and grow when nutrients become available. It has been established that some bacteria can differentiate into spores and fruiting bodies in order to survive starvation (Grossman 1995; Kaplan and Plamann 1996). Several copiotrophie non-differentiating bacteria, on the other hand, have been found to respond to nutrient limitation by forming ultramicrocells and acquire cross- protection to other stresses such as heat shock, ethanol and oxidative stress (Matin 1991; 0stling et aI I993; Spector and Foster 1993; Givskov et al 1994). The cells exposed to starvation respond in an ordered, sequential fashion by expressing genes and proteins thereby allowing them to be relatively less metabolicaIly active while being resistant to an array of environmental challenges. Although the specific regulation of gene expression and the subsequent protein responders is different in the various organisms studied, the hallmark of the starvation response in these organisms includes the formation of viable starvation- and stress-resistant ultramicrocells. A recent study examining the survivat of P. flourescens has clearly shown that bacteria respond to carbon limitation conditions and develop cross-protection to several stress conditions in the soil microcosm (van Overbeek et al 1997). Not surprisingly, exceptions to this model have also been reported. Such is the case for the fish pathogen, Vibrio anguillarum which forms elongated, helical filaments on exposure to starvation. Additionally, starvation-acquired cross-protection to other stresses was lost during long-term starvation (Nelson et al 1997). Another interesting example is that of the oligotroph Sphingomonas sp. strain RB2256 which is greatly resistant to several stress conditions during growth but in general does not acquire additional stress resistance during starvation (Eguchi et al 1996). Hence, while several specific responses to starvation appear to be similar in many strains, a variety of strategies is likely to exist in different bacteria.

The role of small molecular weight extracellular signal molecules in the starvation adaptation programme also warrants further research. Vibrio sp. S14 produces one or several extraceIIular metabolites which appear to be important in the formation of starvation- and stress- resistant ultramicrocells. This represents novel finding wherein the expression of stationary phase phenotype is dependent on small diffusible molecule(s) irrespective of the cell density of the population. In contrast, most


studies report bacteria producing small molecules as bio-monitors o f the cell density of the population (quorum sensors). Moreover, the phenomenon of 'cross-talk' between various extracellutar molecules produced both by prokaryotes and eukaryotes, which is an emerging field o f research, is likely to have significant implications for our understanding of the survival of bacteria in the natural environment.

Acknowledgments

This study was supported by grants from the Australian Research Council, the Centre for Marine Biofouling and Bio-Innovation, The University of New South Wales, Sydney, Australia and the Swedish National Environment Protection Agency (EU Biotechnology Programme).

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cycles of famine and feast: the starvation and outgrowth

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