Journal of Applied Bacteriology Symposium Supplement 1992,73, 49S-57S

Physiology, molecular biology and applications of the bacterial starvation response A. Matin Department of Microbiology and Immunology, Stanford University School of Medicine, CAI USA

1. Introduction, 49s 2. Energy generation during starvation, 50s 2.1 Reserve polymers, 50s 2.2 Ribonucleic acid (RNA), 50s 2.3 Protein, 51s 2.4 Endogenous respiration, 51s 3. Enhancement of scavenging capacity, 51s 3.1 The carbon starvation-escape response, 515 4. Development of general cellular resistance, 5 2 s 4.1 T h e starvation proteins, 5 2 s

5. Starvation gene regulation, 53s 5.1 cst and pex genes-role of cyclic AMP, 53s 5.2 The role of K a t F , 53s 5.3 Role of 03’,54s 6. Biochemical role of stress tolerance-enhancing starvation proteins, 54s 7. Biotechnological uses of starvation promoters, 55s 8. Acknowledgements, 56s 9. References, 56s

1. INTRODUCTION

mechanisms in response to starvation. As a result of expression of starvation genes, these bacteria control degradation of macromolecules as energy and monomer sources, become superior scavengers of the scarce nutrient, and acquire a more resistant endospore-like character (Matin et al. 1989; Matin 1990, 1991). T h e food ecosystem, which is an area of interest in this Symposium, is also impacted by non-growing bacteria. As is discussed below, non-growing bacteria are important in biotechnology in general, including processes concerned with fermentation of food products (see Nout & Rombouts, this Symposium, pp. 136S-147S). Many long-term food processes, such as ripening of cheese, ageing of wines, etc. are also likely to depend on biochemical activities uniquely possessed by non-growing bacteria. For instance, during cheese fermentation, the milk sugar lactose is fully consumed in the first 30 min of fermentation, even though the complete process requires several weeks. Hydrolysis of peptides, lipids and carbohydrates occurs during this time in which the lactococci are likely to be metabolically sluggish. These activities require excretion of specific hydrolytic enzymes and induction of specific uptake systems. It has been shown that Lactococcus lactis does induce starvation proteins when starved for carbon (T. Ubbink, E. Kunje, W. Konings & A. Matin, unpublished). In addition, bacteriocins of lactic acid bacteria, e.g. lactocin B, lactocin 27, diplococcin and lactostrepcins, are produced primarily in the stationary phase. Similarly, aflatoxin is produced by starving Aspergillus sp. (Bills & Kung 1990) (see Moss, this Symposium, pp. 80s-88s). Finally, the enhanced resistance of starving bacteria is relevant to food safety.

Starvation is likely to be the common lot of bacteria in nature. T h e amount of dissolved organic matter in the oceans is between 0.4 and 0.8 mg carbon/l, and most of this is not biodegradable (Morita 1988). According to recent studies, bacterial biomass production in marine and estuarine environments is limited not only by grazing but also by substrate concentration ; in the upper estuary in particular substrate concentration was the limiting factor and the growth rates were probably close to zero (Coffin & Sharp 1987). High-nutrient niches do exist, for example on surfaces and particles where nutrients adsorb, or within animal hosts but, according to the ‘spinning wheel aggregate’ hypothesis of Goldman (1984), such existence probably alternates with long periods of planktonic existence with little or no growth due to nutrient deprivation. T h e mean generation time of a deep sea bacterium was estimated to be 210 d (Carlucci & Williams 1978) and similar low growth rates have been reported in other environments; starvation may be responsible for inducing the virulence apparatus in certain pathogenic bacteria (Matin et al. 1989). Starvation fails to induce dormant, resistant structures such as endospores in a vast majority of bacteria. Although such bacteria have traditionally been considered nondifferentiating as regards starvation, it has become increasingly clear that they activate complex molecular regulatory

Correspondence to: Pro$ A . Matin, Department of Microbiology and Immunology, Sherman Fairchild Science Building, D3J 7, Stanford Unrversity School of Medicine, Stanford, C A 94305-5402, USA.

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2. ENERGY GENERATION DURING STARVATION

A starving cell must require a basal level of energy to stay viable for such processes as maintaining a different ionic mileu than the external environment, repair reactions, and resynthesis of labile macromolecules. Historically, the major query of studies on starvation centred on the identity of endogenous substrates that provided this maintenance energy (Dawes 1976). Implicit in these studies was the hope that it may be possible to correlate survival capability with the concentration of a specific macromolecule in the cell at the onset of starvation.

directly with the PHB content at the onset of starvation, and the polymer was rapidly degraded during starvation. Furthermore, after growth at low D-values, the Spirillum sp. had greater resistance to starvation than a non-polymeraccumulating pseudomonad isolated from the same environment. This advantage disappeared after growth at D = 0.15/h, at which less PHB accumulated. Thus, the prescience of the Spirillum sp. to economize in a harsh environment made it better able to survive starvation than the inherently hardier pseudomonad (Matin et al. 1979).

2.2 Ribonucleic acid (RNA)

2.1 Reserve polymers

Reserve carbon polymers, such as glycogen and poly-Bhydroxybutyric acid (PHB), are accumulated by certain bacteria when cultivated under conditions of carbon excess. Several studies point to the conclusion that such polymers are utilized rapidly during carbon starvation and enhance survival. Micrococius halodenitrificans can accumulate PHB up to 50% of its dry weight under appropriate conditions (Sierra & Gibbons 1962). Such polymer-containing bacteria remain fully viable for 100 h ; in contrast, their counterparts containing only 10Y0 PHB retained 10% viability after just 30 h of starvation. Similar findings have been reported for Alcaligenes eutrophus, Sphaerotilus discophorus and Azotobacter agilis (Dawes 1976). Cellular glycogen has been shown to enhance survival of Escherichia coli. van Houte & Jansen (1970) showed that Streptococcus mitis cells, which can accumulate glycogen up to 37% of their dry weight, survived carbon starvation much better than their glycogen-poor counterparts. After 16 h the former had a viability of 40% and the latter only 0.01 Oh. In bacteria that do not accumulate reserve polymers, cellular RNA probably serves as the major source of energy during starvation and the beneficial effect of the reserve polymers might have been due to sparing of this macromolecule. In general, however, utilization of reserve polymers during starvation proceeds along with that of RNA and at rates comparable with the non-polymer-accumuiating bacteria (Dawes 1976; Matin et al. 1979). Nature being unpredictable and starvation being the rule, especially in oligotrophic environments, why not save an essential nutrient even in times of relative scarcity? This seems to be the strategy followed by a Spirillum sp. adapted to low nutrient environments. When it was grown in a chemostat under carbon limitation, this bacterium accumulated more PHB at low dilution rates (D). At D = 0.15/h, it contained 8% (w/w)PHB, but at D = 0.025/h, the content increased to 18O/;, . Its starvation resistance correlated

The RNA content of the bacterial cell is proportional to growth rate. Even during growth at submaximal rates, several times more RNA appears to be present than required. This macromolecule is degraded rapidly during starvation (Dawes 1976). I n E. colt 20-30% of the cellular RNA was lost within the first 4 h of starvation, with 10% more being lost in the next 20 h. Ribosomal RNA was the preferentially degraded species, and viability correlated with the ribosome content. It was surmised that the culture eventually lost viability because of ribosome depletion (Davis et al. 1986). In Arthrobacter crystallopoites, which forms either rods or spheres depending on the growth rate, the rate of RNA degradation was vastly different in the two forms during starvation. After 30 d of starvation, 85% and 32% of RNA, respectively, had been degraded by the two forms; yet both retained 100% viability during this period (Boylen & Ensign 1970). Initial rapid degradation of RNA during starvation followed by a slower rate has also been reported for other bacteria such as Zymornonas mobilis (Dawes 1976). In other bacteria, however, it appeared to be constant during the first 40 h of starvation. The rate of RNA breakdown during starvation increased the faster the cells had been grown in a chemostat prior to the onset of starvation. For a non-polymer-accumulating bacterium, survival was directly proportional to the RNA content at the onset of starvation. Thus, in this case both the content and rates of RNA breakdown correlated with starvation survival capacity (Matin et al. 1979). RNase activity increases several-fold upon starvation and this is probably because of activation of pre-existing RNases since inhibitors of protein synthesis do not interfere with this phenomenon (Matin et al. 1989). Polysomes are rapidly broken down into individual ribosomes upon starvation. RNase I further degrades the 70s monosomes into 30s and 50s subunits. Two enzymes, RNase I1 and polynucleotide phosphorylase, degrade 16s rRNA. The ribosomal subunits are then dissociated into protcins and nucleotides, and the proteins become attached to the cell membrane (Kaplan & Apirion 1975).

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2.3 Protein

The rate of protein degradation increases substantially at the onset of starvation. There is also net reduction in the cellular protein content. In E. coli net protein degradation occurs primarily after RNA breakdown (Dawes 1976), but in other bacteria concurrent degradation has been reported. Thus, in Bdellevibrio bacteriovorus ca 14% of the protein is decreased within 6 h of starvation and this degradation occurs concomitantly with net RNA degradation (Hespell et al. 1974). I n the rod forms of A. crystallopoites, 45% of the protein had been degraded by 30 d of starvation compared with only 20% in the spheres. As pointed out above, both forms maintained 100% viability during this period (Boylen & Ensign 1970). Other bacteria in which net degradation of protein during starvation has been documented include Z. mobilis, Lact. lactis and Pseudomonas aeruginosa. That the amino acids generated by this protein breakdown serve as a source of energy is suggested by the finding that ammonia is produced by many starving bacteria (Dawes 1976). Conversion of pre-labelled cellular protein to CO, during starvation has also been demonstrated (Bockman et al. 1986). In E. coli nine soluble endoproteolytic activities which degrade protein substrates have been described. Protease I11 degrades polypeptides of less than 7 kD. Proteases IV and V are membrane-associated, and proteases Ion and T i are ATP-dependent. It is not known which of these proteases are involved in protein degradation, nor whether their induction or activation account for increased proteolysis in starvation. Activation of pre-existing proteases is supported by the finding that protein substrates in vitro can allosterically increase protease activity (Waxman & Goldberg 1986; Miller 1987; Hwang et al. 1988). The stringent response is also involved in the control of proteolytic activity in an unknown manner.

2.4 Endogenous respiration

The utilization of cellular constituents during starvation is manifested by a basal level of endogenous metabolism, which can be measured by oxygen consumption, CO, evolution or other means. A number of studies that sought to correlate the levels of individual macromolecules to survival have led to the conclusion that it is not the absolute level of individual cellular constituents that determines longevity, but the control that bacteria can exercise over their degradation during starvation. Thus bacteria that are able to utilize endogenous macromolecules at a slow rate appear to have an advantage over those less able to control this process. Thus, the ‘champion starvation survivor’ (Dawes 1976), A. crystallopoites, has a markedly lower endogenous

respiration rate than most other bacteria. Bdellevibrio, which loses viability rapidly, has a high rate of endogenous respiration. Two factors might be decisive: an inherently low maintenance energy requirement, and the capacity to reduce metabolic rate to match this requirement during starvation.

3. ENHANCEMENT O F SCAVENGING CAPACITY

When they are subjected to the dearth of a nutrient bacteria enhance their potential for scavenging that nutrient by acquiring a higher affinity for the missing nutrient and/or expanding their metabolic potential to use additional sources. T h e genetic basis of enhanced assimilation capacity for nitrogen, phosphorus and iron have been well elucidated. By contrast, the genetic basis of the carbon starvation-escape response is less well understood. I will discuss here only the latter. For information on other nutrients, other reviews may be consulted (Kustu et al. 1986; Bagg & Neilands 1987; Wanner 1987; Matin et al. 1989).

3.1 The carbon starvation-escape response

The available information focuses mainly on the phenotypic aspects. It is not known whether sensors and transcriptional activators and minor sigma factors co-ordinate this response. Phenotypic studies involving bacteria cultivated under carbon limitation in the chemostat and starvation of marine bacteria indicate an increase in the concentration of substrate-capturing enzymes and those of intermediary metabolic pathways, as well as induction of high affinity uptake systems for carbon substrates. For example, when two strains isolated from fresh water were cultivated at different dilution rates under lactate limitation, the concentration of lactate dehydrogenase (the substrate-capturing enzyme), isocitrate dehydrogenase (energy-generating pathway representative) and glucose-6-phosphate dehydrogenase (biosynthetic pathway representative) increased up to 10-fold when growth occurred at a steady-state lactate concentration of ca 2 pmol/l as opposed to growth at 100 pmol/l and over (Matin 1979). A Vibrio sp. possesses high as well as low affinity transport systems for mannitol, glucose and glutamate. T h e K,,, for mannitol uptake was ca 7 pmol/l during the first 5 weeks of starvation, but then decreased to ca 0.2 pmol/l (Kjelleberg et al. 1987). More recent studies involving the use of two-dimensional gel electrophoresis and reported fusions to starvation genes have revealed a large number of proteins that are induced

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by carbon starvation and require cyclic AMP (CAMP) for this induction. These proteins, termed the Cst proteins (Matin 1991), are believed to be primarily concerned with escape from carbon starvation. A cstA mutant was impaired in peptide utilization and it is likely that this cst locus is concerned with genes involved in peptide transport. Induction of cstA thus expands the metabolic potential of E. coli thereby enhancing its chances of escaping starvation (Schultz & Matin 1991).

4. DEVELOPMENT OF GENERAL CELLULAR RESISTANCE 4.1 The starvation proteins

A major role of protein degradation in starvation is the provision of amino acids for the synthesis of proteins conducive to starvation and stress survival. T h e peptidase-deficient mutants of E. coli are impaired in starvation survival. They are defective in amino acid generation because protein degradation leads only to formation of diand tripeptides. Consequently, protein synthesis is greatly impaired in these mutants during starvation which accounts for their poor survival. Inhibition of protein synthesis in starving wild type E. coli also compromises long-term survival, and the intensity of the effect depends on how soon after starvation protein synthesis is inhibited. Thus, proteins synthesized in early starvation are essential for effective long-term survival (Matin 1991). It was shown that cryptic growth did not occur in these experiments. Cryptic growth refers to the phenomenon in which a part of the starving culture multiplies at the expense of nutrients released by lysis of dead cells (Reeve er al. 1984). Two-dimensional gel electrophoresis studies have provided direct evidence for the synthesis of starvation proteins (Groat et al. 1986; Spector et al. 1986; Nystrom et al. 1990). Some 55, 35 and 47 polypeptides were induced in E. coli during starvation for carbon, phosphorus or nitrogen, respectively. A detailed analysis of these proteins has revealed that: ( 1 ) the starvation protein synthesis lasts for 2 4 h depending on the preceding growth conditions; (2) each starvation regimen has its own individual signature of protein induction, but a core set of some 15 proteins is synthesized by starving E. coli when starved for several individual nutrients ; (3) different starvation proteins fall into different temporal categories indicating sequential gene induction. The synthesis of the early proteins commences within minutes of the start of starvation, whereas that of the late proteins starts when bulk protein synthesis has declined to very low values; and (4) many of the starvation

proteins, particularly those constituting the core set, are common with proteins synthesized by E. Cali during heat shock, oxidative or osmotic stress (Groat et al. 1986; Schultz et al. 1988; Spector et al. 1986; Jenkins et al. 1988, 1990; Nystrom et al. 1990). As might be expected from the overlap of starvation proteins with those of other stresses, starved E. colt cells acquired resistance to lethal doses of heat, H,O, , hyperosmosis, acid and disinfectant agents. The phenomenon is illustrated for heat stress resistance. Maximal protection occurs within 4 h of starvation (Fig. 1). T h e pattern of induction of resistance to other stresses is very similar (Matin 1991). I t has also been shown that E. coli and Salmonella typhirnuriurn exposed to an adaptive dose of H,O, develop increased resistance to a lethal dose of heat (Christman et al. 1985; VanBogelen et al. 1987). Starvation genes fall into several different regulatory classes. Mutants have been isolated that synthesize only a

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Fig. 1 Induction of thermal resistance in Escherichia colt during glucose starvation. A wild type K-12 culture at 37°C was challenged at 57°C 0, during exponential growth or at A, 1 h ; A, 2 h; lJ,4 h or 24 h after glucose depletion from the medium. A control experiment was also performed, in which chlorarnphenicol (100 pg/ml) was added 20 rnin after the onset of starvation and the culture was allowed to starve for 4 h before heat challenge (a).On the y-axis, 100% is equivalent to ca 3 x lo3 cells/ml. Reproduced by permission from Jenkins et al. (1988)

MOLECULAR BIOLOGY OF S T A R V A T I O N 53s

subset of stress proteins upon starvation, and their resistance pattern has excluded or implicated certain proteins in stress resistance. This will now be discussed.

cAMP also has a role in sensing cell density or other intercellular communication. N o evidence, however, as yet supports such a possibility.

5.2 The role of KatF 5. STARVATION GENE REGULATION

5.1 cst and pex genes-role

of cyclic AMP

Of the genes induced and proteins synthesized by carbon starvation, two-thirds require cAMP for their induction. Cyclic AMP is synthesized by the enzyme adenylate cyclase (encoded by cya) and it exerts its regulatory role in bacteria by binding to the cAMP receptor protein (CRP; encoded by crp). In a Acya or Acrp genetic background, only onethird of these proteins are induced upon starvation. Most of these CAMP-independent proteins belong to the core set, although some are unique to carbon starvation. The CAMPdependent and -independent starvation genes are termed cst and per genes, respectively. T h e Acya or Acrp strains of E. colz develop the starvation-mediated general resistance in the same way as the wild type (Matin 1990). Thus, the cst gene induction does not have a role in this phenomenon. As discussed above, the Cst protein induction is thought to be concerned with enhancing the carbon scavenging capacity of the cell (Matin et al. 1989). The molecular regulation of four cst genes has been characterized in some detail and reviewed elsewhere (Matin 1990). The cstA gene is transcribed during starvation from three sites, upstream of each of which there is an Eo7'-10 sequence. Eighteen nucleotides upstream of the strongest start site, a sequence resembling the consensus CRP binding site was found, whose partial removal abolished transcription from all start sites (Schultz & Matin 1991). The cstA gene induction appears to be due entirely to the increased cAMP levels of starved bacteria. I n contrast, induction of other cst genes requires additional effector(s). The identity of the latter is not known, but it is present in the culture fluid of the stationary phase cells. Whether it is synthesized only in the stationary phase, or accumulates during the exponential phase, is not yet known. The similarity of this class of cst genes to the bioluminescence (lux) operon induction is noteworthy. The latter also requires cAMP plus a molecule, called the autoinducer, which accumulates in the culture medium. I t is postulated that the autoinducer is a device for sensing cell density (Slock et al. 1990). By analogy it appears possible that the extracellular regulators of cst genes have a similar role. Almost all (over 99.9%) of the cAMP made by E. coli grown at several dilution rates in a chemostat was excreted into the medium (Matin & Matin 1982). It is possible that extracellular

KatF is a putative cr factor (Mulvey & Loewen 1989) which is essential for the expression of two enzymes concerned with oxidative stress protection in the stationary phase, namely hydroperoxidase (encoded by katE gene) and exonuclease I11 (encoded by xthA gene). Mutants deficient in the katF gene fail to induce some 32 carbon starvation proteins, including six previously identified as pex. These mutants are impaired in starvation survival under a variety of conditions, i.e. carbon starvation under aerobic or anaerobic conditions, as well as nitrogen starvation. Many of the starvation proteins not synthesized by the mutant overlap with other stress proteins. For example, nine osmotic stress/starvation proteins are not induced in the starved katF mutant, and it fails to develop starvation-mediated resistance to hyperosmosis. Similarly, several oxidation and heat shock proteins are not induced, and starvation fails to confer enhanced resistance to these stresses on a katF mutant (McCann et al. 1991). Nor is starvation alone in failing to elicit an adaptive response from the katF mutant. Thus, exposure to sublethal doses of heat, hyperosmosis and oxidative stress, which make the wild type resistant to these individual stresses, fails to protect the mutant (McCann et al. 1991; McCann & Matin, unpublished). regulated heat shock proteins were thought to be sufficient in thermal protection in E. colz, until the studies of VanBogelen et al. (1987). These workers used the tac promoter to control cr32 synthesis, and induced regulated heat shock proteins in E. coli without heat shock and found no thermal protection. Similar experiments need to be done with a katF mutant to determine if KatF-regulated proteins are sufficient to confer multiple resistances on E. coli. Nevertheless, it is clear that the katF-controlled regulon is a major mechanism in managing the stress responses of E. coli. Both katF and xthA genes possess unique -35 and - 10 sequences [GTTTAGC and ACGTCC, respectively (Ossowski et al. 1991)], which may be uniquely recognized by the putative EaKatF.I t remains to be seen how many of the genes that fail to induce in the starved katF mutant possess such a sequence. Recently, we have found a similar sequence in the promoter of the KatF-regulated pexA gene (Rhie et al., unpublished). Many of the KatF-regulated pex genes are negatively effected by cAMP in that they are hyperinduced in a Acya or Acrp background. The reason for this is not known. KatF also regulates some of the cst genes which appear to be Ea7'-transcribed as a class.

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These may possess multiple promoters recognized by different species of RNA polymerase holoenzymes. T h e fact that a given katF-regulated protein is induced by one stress but not by another, even though the latter causes induction of other katF-regulated proteins, suggests involvement of additional regulatory mechanisms specific to individual stresses. Whether KatF levels are modulated in response to stresses is not known. I t is clear that the molecular mechanisms underlying KatF-mediated regulation are multifaceted and will be the subject of much future interest.

5.3 Role of c12 0 3 2is a minor o factor that was identified in the context of heat shock. Many of the genes that are induced upon heat shock possess unique -35 and - 10 sequences (consensus: CCCCC and CCCC, respectively) that are recognized by Ea3*. 03’ levels increase during starvation and work with a mutant deleted in rpoH (the gene that encodes 032) showed that these increased levels account for starvationmediated induction of at least three proteins, namely DnaK, GroEL and HtpG. The rpoH deletion strain is impaired in starvation survival, but can develop starvationmediated cross-protection to heat and oxidation. Thus, these three genes appear to play a role in starvation survival, but not in starvation-mediated resistance to other stresses (Jenkins et al. 1991). It has been shown that the regulatory region of 03’ is complex, containing sequences that are transcribed by different species of RNA polymerase holoenzymes. It also contains a CRP site (Nagai et al. 199O), which may account for the enhanced levels of o J 2 during carbon starvation. It is not yet known, however, whether these increased levels result from transcriptional or post-transcriptional regulation.

6. BIOCHEMICAL ROLE OF STRESS TOLERANCE-ENHANCING STARVATION PROTEINS

The identity and biochemical function of most of the proteins conferring starvation-induced general resistance are unknown. The only cxceptions are DnaK, GroEL and HtpG proteins that are involved in resistance to starvation, and hydroperoxidase 11, which is partly responsible for starvation-mediated oxidation resistance. T h e last mentioned enzyme is a hexamer of 93 kDa subunits, and is likely to owe its protective role to its activity as a catalase. The other three proteins are among the general stress protein homologues of which are synthesized by all organisms and exhibit a remarkable conservation through evolution. For example, DnaK of E. colz bears over 50%

homology to its eukaryotic counterpart (HSP70) in yeast, Drosophila, and mammals. Many of the stress proteins appear to have an overlapping function, and yet may play unique roles. For example, GroEL and GroES proteins are required for growth of E. coli at normal physiological temperatures, whereas DnaK may be more important at higher temperatures (Kusakawa & Yura 1988). The major functions of stress proteins appear to be protein folding, protein cleavage and assembly and disassembly of multiprotein structures. T h e GroE proteins are necessary for proteolytic cleavage of phage proteins, which are synthesized initially as long strings, apparently by generating the conformation required for proteolytic activity. HSP70 is required for translocation of proteins into organelles. I n an in vitro system, prepro c1 factor, a precursor of yeast sex pheromone, cannot translocate to endoplasmic reticulum (ER) unless HSP70 and ATP are provided. T h e counterpart in z~zvoexperiment relied on genetic manipulation of yeast so as to regulate the amount of HSP7O in the cell. With decreasing amounts of this protein in the cell, there was an increasing build-up in the cytoplasm of proteins destined for the ER (Deshaies et al. 1988). Similarly, the B-cell immunoglobulin-binding protein, BIP, which had been known to be essential for the correct assembly of heavy and light chains of antibodies in the ER, was subsequently identified as HSP70. Recently, evidence has been obtained for the role of DnaK in the correct assembly of proteins in E. colz. When over-producing proteins [e.g. human growth hormone (HGH)] this bacterium can form ‘inclusion bodies’ that represent denatured precipitated proteins. If, however, DnaK concentration is increased, particularly before overproduction of HGH, there is a marked decrease in inclusion body formation and increase in soluble H G H (Blum et al. 1991). Thus, a consensus is emerging that stress proteins like DnaK (HSP70) are essential for correct folding of all proteins. They combine with the nascent polypeptides to keep them from folding prematurely. Under conditions of stress, the elongation of the polypeptide chain is likely to proceed more slowly and this may account for the increased need for some of the stress proteins and their protective role. A more direct role for this protection in actually rescuing proteins denatured by stress has also been postulated (Pelham 1986). I n denatured proteins, hydrophobic residues are likely to become exposed which may be the sites recognized by stress proteins. By combining with these residues, the stress proteins would disaggregate the clumps of denatured proteins while, using the energy from ATP, bringing about their unfolding. This would afford the denatured cellular proteins another chance to fold in their correct configuration. However, there is no direct evidence in support of this postulate. If anything, its central tenet would appear to be negated by the fact that

MOLECULAR BIOLOGY O F STARVATION 55s

HSP70 binds avidly to both hydrophobic and hydrophilic proteins.

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7. BIOTECHNOLOGICAL USES OF STARVATION PROMOTERS

For many purposes it is useful to have expression of a desired biochemical activity in slowly or non-growing bacterial cells ; for example, in dense microbial fermentations. Dense microbial aggregates provide powerful catalytic activity for rapid conversion of substrate to product, but they cannot be kept in an active growth state because of diffusivity problems and space constraints. Furthermore, their continued growth diverts nutrients into cell biomass rather than into product formation. A non-growing dense cell configuration preferentially expressing biochemical activities of interest would therefore be useful in bioprocessing. Strains have been constructed in which the HGH gene has been spliced behind the cstA promoter. These strains produce HGH primarily in the stationary phase (Matin 1990). In many food fermentations, for example ripening of cheese, it is probable that non-growing bacterial cells are involved. Engineering of such cells so that desired biochemical activities are expressed at a high level during the ripening process would therefore need to depend on the use of starvation-type promoters. The expression of enzyme activities in slowly growing bacteria is also of interest in bioremediation. Indigenous populations in contaminated sites, such as estuaries, frequently possess the genetic potential to convert the toxic materials to innocuous end-products. However, since expression of these genes is generally driven by growthrelated transcription elements, this potential is of little practical use in situ because of low nutrient concentrations and metabolic activity. Therefore, starvation promoterdriven tmo expression systems in E. coli have been constructed ; tmo encodes toluene monooxygenase activity, which can degrade trichloroethylene (TCE) and phenol, the two common environmental pollutants (Little et al. 1991). The recombinant strain exhibits a strong activity for phenol and T C E degradation in the metabolically sluggish state. In contrast, the tac-driven system is virtually inactive (Fig. 2); the tar promoter in the presence of IPTG is powerfully expressed in growing cells. Clearly, totally starved cells cannot express any biosynthetic activity for long since energy depletion and loss of viability will eventually ensue. Thus, if starvation/stress promoters were expressed only in completely starved cells, their utility would be limited by their short activity span. Most of the starvation promoters so far examined, however, are powerfully induced also at very slow growth rates. This is illustrated for a cst: :lac2 fusion cultivated at different

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Fig. 2 Phenol degradation by non-growing recombinant strains of Escherichia coli, containing the tmo gene under the control of 0 ,a growth promoter (tar) or 0, a stress promoter (groEL). IPTG was added to the suspension of strain containing the tac: :tmo plasmid. Modified from Little et al. (1991)

dilution rates in a chemostat under glucose limitation. There is increasing activation of the promoter with decreasing D-values. At 0.05/h (ca 14 h generation time), the lowest D-value examined, the steady state level of the expression was nearly 100-fold higher than at near pmax (Fig. 3). Thus, these regulatory elements can provide a means of obtaining a sustained high level expression of biochemical activities of interest in very slowly growing bacteria.

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Fig. 3 Effect of dilution rate on the expression of an Escherichia coli cst promoter as monitored by B-galactosidase production. The limiting nutrient was glucose. S. Schippa and A. Matin,

unpublished

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8. ACKNOWLEDGEMENTS T h e unpublished work cited from this laboratory was supported by NIH grant 1RO1-GM42159, Western Region Hazardous Substances Research Center grant RPA R815738, and the State of California Competitive Technology grant C88-142. T h e author thanks Jill Looper for expert secretarial help.

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