INTRODUCTION

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In the natural environment, chemoorganotrophs such as Escherichia coli obtain both their carbon and energy from organic matter released by other cells. The mechanisms of organic nutrient release are variable, ranging from regulated extrusion of metabolic end products to release as a result of death and lysis of donor cells (3, 21-23, 34). However, the actual bioavailability of carbon in nature is low due to intense competition (22, 23). As a result, natural microbial populations spend the majority of their lives under starvation stress, interspersed with sporadic and short-lived periods of growth as nutrients become available.

Our laboratory uses carbon-starved cultures of E. coli as an experimental model to understand the processes of survival and evolution in natural microbial populations. E. coli can survive extended periods of starvation. In aerated rich medium (Luria-Bertani [LB] broth), E. coli ceases growth due to carbon limitation (37). During the first several days of starvation, the population loses 90 to 99% of the viable counts (40). However, the viable counts nearly level off after these first few days, and populations can survive for several years in this spent LB medium aerated at 37°C without further addition of carbon (7, 8). As the cultures consume exogenous carbon during exponential growth, the biomass is the most likely source of carbon during extended survival, which becomes available when the cells die.

While the overall population of stationary-phase E. coli cultures may be considered starved in that there is no net increase in biomass, there are subpopulations that are clearly not starved, as they are able to grow as a subculture and take over the population (8, 38-40). These subpopulations consist of mutants with enhanced fitness during starvation. The ability to grow during starvation has been termed the growth advantage in stationary phase (GASP) phenotype (38). Studies on cultures starved for extended periods demonstrate that the GASP phenomenon is continuous: multiple rounds of population takeovers occur throughout the starvation period (7, 40). Interestingly, as the cultures age, they increase in diversity, as several genetically distinct subpopulations coexist (7).

The first mutation conferring the GASP phenotype after growth in rich media was identified as an allele of rpoS (40), a gene whose product, ^S, is responsible for the regulation of many genes during starvation stress (12). Transduction of the GASP allele of rpoS (rpoS819) into an otherwise wild-type strain was sufficient to confer the GASP phenotype (40). The rpoS819 allele is a 46-bp duplication at the 3' end of the gene, which results in a replacement of the last four residues in ^S with 39 new amino acids. Expression of two ^S-dependent genes, katE (25) and bolA (4, 15), are both reduced in the rpoS819 strain (40), indicating a reduction of function in this allele. The physiological basis for the fitness gain of the rpoS819 mutation is not yet known.

The purpose of our investigation was to understand how GASP mutations alter cell physiology to provide fitness gains in stationary phase. To this end, we sought to identify and characterize new GASP mutations. ZK1141, an isolate from an aged culture of the rpoS819 strain, was capable of outcompeting its rpoS819 parent, indicating that additional GASP mutations accumulated in this strain (38). In this study, we have demonstrated that the ZK1141 strain has acquired three new GASP mutations, each of which can confer the GASP phenotype on the rpoS819 parent. Each of these newly identified GASP alleles, as well as the rpoS819 allele, increased starvation survival fitness in an additive manner. Each of these four GASP alleles also conferred growth advantages on amino acids as the sole sources of carbon and energy. Similar to the competitive fitnesses, these growth phenotypes were additive.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains. The E. coli strains used in this study are listed in Table 1.

Media and growth conditions. All experiments were performed at 37°C, except where noted. The media used in this study have been previously described (19). M63 minimal medium was supplemented with 1 µg of thiamine per ml and 1 mM MgSO₄. All amino acids used in this study were of the L configuration. Where appropriate, LB plates were supplemented with streptomycin (25 µg/ml), nalidixic acid (20 µg/ml), tetracycline (15 µg/ml), kanamycin (50 µg/ml), or chloramphenicol (30 µg/ml). All chemicals were from Sigma. Optical density (OD) was monitored with a Spectronic 20D+ Spectrophotometer (Milton Roy).

Genetic techniques. Phage P1vir transduction and Hfr conjugation using the Singer et al. (31) strain collections were performed as described elsewhere (19). Insertional mutagenesis with mini-Tn10 transposons from the vectors NK1323 (Tc^r), NK1316 (Kan^r), and NK1324 (Cm^r) was performed as described previously (13).

Construction of GASP strains. Because incorporation of new genetic markers can alter the fitness of bacteria, we constructed our strains such that the final strains differed from the parental strains only by the allele(s) of the GASP loci. This was achieved by first bringing an auxotrophy mutation or the streptomycin-sensitive (Sm^s) allele of rpsL that mapped near the GASP loci (see Results) into the recipient by P1vir transduction. We could then cotransduce the GASP alleles with P1vir grown on ZK1141 or ZK126 into these strains by selecting for prototrophy or Sm^r, and then testing among those transductants for the cotransduction of the GASP allele, by assaying directly for the GASP phenotype or another physiological phenotype where appropriate (see below).

Stationary-phase competitions. Competition experiments were adapted from those of Zambrano et al. (40). For competitions in LB, initial cultures were inoculated from frozen glycerol stocks into 3 ml of LB and grown overnight. These were then subcultured 1:100 into fresh LB and incubated for 24 h before being mixed for the competitions. The two populations were monitored by serial dilution in M63 medium and plating on minimal salicin and minimal glucose-plus-valine plates. We verified that the majority of the population remained prototrophic (and hence detectable on the selection media) by comparing the counts on the selection media with those on LB. In no case did we observe a difference in total viable counts on minimal and rich media. For competitions in M63-serine (0.5%)-isoleucine (0.03%)-valine (0.03%)-leucine (0.03%)-NaCl (0.5%), colonies were inoculated from an LB plate into the defined medium, and the cultures were incubated until they reached stationary phase (1 to 4 days). Cultures of strain ZK2618 (trpB::Tn10) were also supplemented with 0.004% tryptophan to facilitate growth. The ZK1141 and ZK2618 cultures were washed in M63 medium before inoculating as a 1:10,000 minority into the ZK820 cultures. The two populations were monitored by serial dilution in M63 medium and plating on LB-streptomycin and LB-nalidixic acid.

Molecular techniques. The DNA flanking the mini-Tn10Cm^r transposons was determined by using an arbitrary PCR-based protocol (5), with the modifications described by Pratt and Kolter (28). PCR products were subjected to sequence analysis by the Micro Core Facility, Department of Microbiology and Molecular Genetics, Harvard Medical School, and the sequences were compared with the GenBank DNA database by using the BLAST program (1).

	RESULTS

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Isolation and mapping of three new GASP loci from an aged rpoS819 strain. Our approach to investigate the physiology of the GASP phenomenon was to identify and characterize several mutations able to confer the GASP phenotype. That many rounds of GASP takeover occur in starved cultures of E. coli (7, 40) implies that the survivors have acquired multiple GASP mutations. As we were interested in exploring potential genetic interactions of accumulated GASP mutations, our strategy was to isolate and investigate the GASP mutations of a single mutant survivor (ZK1141) from a culture that had undergone several rounds of population takeover.

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Mapping the G_II GASP loci. During our investigations of strain ZK1141, we discovered that it has two phenotypes that its ZK819 parent lacks: mucoid growth on glucose at 30°C (but not at 37°C) and an enhanced sensitivity to the amino acid serine. Serine is a competitive inhibitor of homoserine dehydrogenase I, and high levels of intracellular serine result in isoleucine starvation (9, 10). We scored relative serine sensitivities by streaking the strains on an M63-glucose plate toward a filter disc soaked with 10% serine placed in the center of the plate and determining the relative sizes of the growth inhibition zones. Instrumental in the mapping of the sgaB locus was the discovery that the sgaB GASP allele is responsible for both the mucoidy and serine sensitivity phenotypes of ZK1141. The sgaB mutation was mapped to min 20 by using the Hfr and P1 mapping sets (31); the sgaB mutation was 95% linked to the zbj-3110::Tn10Kan^r marker of CAG18528, located at min 19.8 (2, 31).

The GASP phenotypes of three new GASP loci: sgaA, sgaB, and sgaC. Having isolated and mapped three GASP alleles of strain G_II, we wanted to test whether each of these alleles alone could confer a selective advantage over the G_I parent during stationary phase. We assayed for the GASP phenotype by mixing 24-h-old cultures of the two strains in question and monitoring changes in each population by viable count assay. The two populations were distinguished because they carry different neutral markers. Marker neutrality was confirmed empirically by switching the markers between the strains and performing all mixes reciprocally. Viable counts of each population are determined by titering the culture on the two relevant selection plates. Previous reports suggested that the rpsL allele conferring Sm^r and the gyrA allele conferring Nal^r are neutral in stationary-phase competitions in E. coli (7, 40). We confirmed that the Sm^r and Nal^r markers are neutral during extended starvation. However, during the first 4 days of competition of a 1:1 mix (see below), viable counts were consistently 2- to 10-fold higher for the Sm^r strain than for the Nal^r strain, although the Sm^r counts eventually dropped to equal those of the Nal^r population. Since the first 4 days of the competitions in this study were critical, we differentially marked the ZK819 (Sm^r) strain with two new markers that remain neutral throughout the competition: valine-resistant growth on glucose (Val^r) and the ability to grow on -glucosides (Bgl⁺). The Val^r and Bgl⁺ markers were isolated as spontaneous mutations conferring the ability to grow on M63-glucose-valine or M63-salicin plates, respectively. Unless otherwise noted, this pair of markers was used for all competitions described below.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   The Valr and Bgl+ markers are selectively neutral in stationary phase. One-day-old cultures of the Valr (ZK2552) () and Bgl+ (ZK2553) () derivatives of the GI mutant (ZK819) were mixed 1:1 and cocultured. Viable counts were assayed on M63-glucose-valine or M63-salicin plates. Neither strain was outcompeted in four competitions.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   The GII alleles of ZK1141 confer the GASP phenotype. Into a 1-day-old culture of the GI mutant (ZK2552) () was inoculated as a 1,000-fold minority of a 1-day-old culture of the GII strain (ZK2555) (A), the GI sgaA strain (ZK2561) (B), the GI sgaB strain (ZK2559) (C), or the GI sgaC strain (ZK2563) () (D). Asterisks indicate that viable counts fell below detectable levels (<103 CFU/ml). The patterns of GASP takeovers were identical in six replicate mixtures, including ones where the selectable markers were switched between competing strains.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   The GII alleles confer a competitive advantage to GI cells. A 1-day-old culture of the GI mutant (ZK2552) () was mixed 1:1 with a 1-day-old culture of the GII strain (ZK2555) (A), the GI sgaA strain (ZK2561) (B), the GI sgaB strain (ZK2559) (C), or the GI sgaC strain (ZK2563) () (D). Asterisks indicate that viable counts fell below detectable levels (<103 CFU/ml). The patterns of GASP takeovers were identical in six replicate mixtures, including ones where the selectable markers were switched between the competing strains.

The three G_II GASP mutations are necessary and sufficient for the G_II GASP phenotype. Our selection method identified three new distinct GASP loci on the chromosome of G_II: sgaA, sgaB, and sgaC. To determine whether there are additional GASP mutations in G_II, we examined whether the three GASP mutations identified so far were both necessary and sufficient for the G_II GASP phenotype. Our first approach was to compete the G_II mutant with a constructed G_I sgaA sgaB sgaC mutant. Neither strain had a competitive advantage when competed in a 1:1 mix (Fig. 4). Furthermore, the G_II mutant was unable to grow when inoculated as a 1:1,000 minority into a culture of the G_I sgaA sgaB sgaC (data not shown). These results demonstrate that the G_II strain lacks any additional GASP mutations that would confer a competitive advantage over the constructed strain. Additionally, the G_I sgaA sgaB sgaC strain grew immediately and completely displaced the G_I strain as the majority when inoculated as a 1:1,000 minority into the G_I culture (data not shown); this GASP phenotype was indistinguishable from that of the G_II strain (Fig. 2A). The results from these three different competition experiments thus demonstrate that the sgaA, sgaB, and sgaC mutations are sufficient for the G_II GASP phenotype.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   The sgaA, sgaB, and sgaC alleles are sufficient for the GII GASP phenotype of ZK1141. A 1-day-old culture of the reconstructed GII strain GI sgaA sgaB sgaC (ZK2564) () was mixed 1:1 with a 1-day-old culture of the GII strain (ZK2554) (). Neither strain was outcompeted in eight competitions.

GASP mutants obtain nutrients from dying cells in a chemically defined medium. Because growth in LB ceases due to carbon limitation (37), it has been assumed that the GASP mutants obtain the nutrients required for growth from the dying majority population. To demonstrate directly that the dying cells release nutrients which the GASP mutants can utilize during growth, we identified a chemically defined medium in which the G_II GASP strain could grow and outcompete the G_I strain. Figure 5A shows the GASP phenotype of the G_II strain versus G_I after both cultures were grown to stationary phase in M63 salts medium supplemented with serine (0.5%), isoleucine, valine, and leucine (0.03% each), and NaCl (0.5%). As in LB, the G_II strain grew rapidly as a 1:10,000 minority to take over the population. Like the prototroph, a tryptophan auxotrophic derivative of the G_II strain (trpB::Tn10) was able to grow and take over the G_I minority (Fig. 5B). This finding indicates that the G_II strain can scavenge enough tryptophan to meet its growth requirement during the population takeover. As no exogenous tryptophan was supplied by the medium, the only remaining source of tryptophan is the cells of the dying majority population. This is the first evidence that the GASP mutants can scavenge nutrients released by the dying cells during carbon starvation.

View larger version (12K): [in this window] [in a new window]   FIG. 5.   GASP in a chemically defined medium. Stationary-phase cultures grown in M63-serine (0.5%)-isoleucine (0.03%)-valine (0.03%)-leucine (0.03%)-NaCl (0.5%) (plus tryptophan [0.004%] for the GII trpB::Tn10 strain [ZK2618]) were mixed 1:10,000 to assay the GASP phenotype. Both the GII strain (ZK1141; ) (A) and the tryptophan auxotrophic GII trpB::Tn10 strain (ZK2618; ) (B) express the GASP phenotype as a minority versus the GI strain (ZK820; ). The patterns of GASP takeovers were identical in four competitions.

The three G_II GASP alleles and the rpoS819 allele confer faster growth on amino acids. We reasoned that the primary selective force acting on carbon-starved cells is the ability to utilize the carbon released by the dying majority population. Therefore, we asked whether the GASP mutations confer faster growth on carbon sources that may resemble the nutrients released by dying cells. The three G_II GASP mutations were tested in the G_I background, as it was from this background that G_II was selected. We also tested the growth phenotypes of the G_I GASP mutation, rpoS819, by comparing its growth rate with that of the G_I strain carrying the rpoS⁺ allele (this is essentially a G₀ strain, as the rpoS819 mutation is sufficient to confer the G_I GASP phenotype of ZK819 [38]). We first assayed growth on LB, a rich medium containing many of the building blocks, vitamins, and energy sources necessary for growth. The growth rates on LB were indistinguishable between the different mutant strains (data not shown).

	DISCUSSION

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Previous work identified an allele of rpoS, rpoS819, as a mutation that can confer the GASP phenotype on E. coli (40). Genetic analysis of an isolate (G_II) from a starved culture of the rpoS819 GASP strain (G_I) has revealed three new GASP mutations: sgaA, sgaB, and sgaC. All four GASP mutations of this isolate map to different regions of the chromosome, suggesting that there are many loci that when mutated can confer fitness advantages in stationary phase.

As each of the three new GASP mutations acquired by the G_II strain can confer a GASP phenotype on the G_I parental strain, it is most likely that they were acquired as a result of three successive GASP takeover events. G_II was isolated from a culture starved for a total of about 2.5 weeks, which suggests that in our experimental system population takeovers during starvation can happen faster than once per week. The fact that the three G_II GASP mutations are necessary (and sufficient) for the G_II GASP phenotype demonstrates directly that the acquisition of multiple GASP mutations can provide successively higher fitnesses for starved bacteria. The continual accumulation of GASP mutations by cells within the surviving population can account for the multiple rounds of population takeovers observed in starved cultures (7, 40). Our results thus support the growing body of evidence that starved populations are highly dynamic and undergo frequent population takeovers as a result of fitness differences among the competing subpopulations (7, 8, 40).

A major purpose of our investigation was to understand how GASP mutations alter cell physiology to provide fitness gains in stationary phase. Previous studies have demonstrated that fitness gains during conditions of limited substrate availability in chemostats or selections for growth on novel substrates were manifested as increases in catabolic potential for those substrates (11, 17, 20, 24, 33, 36). In our system, we have observed that dying cells in stationary-phase cultures release nutrients (e.g., tryptophan) that can be utilized by the growing GASP mutants, and we hypothesized that the GASP mutants selected are those that outcompete their parents for these limited substrates. We observed a direct correlation between relative GASP fitness and relative ability to catabolize amino acids, which are likely to be the most abundant nutrients released by dying cells (26). The G_I GASP mutation rpoS819 and the three G_II GASP mutations sgaA, sgaB, and sgaC all confer higher growth rates on an amino acid mixture (Casamino Acids). To our knowledge, this is the first report that mutations in rpoS can alter amino acid catabolism during exponential growth, extending previous observations that wild-type ^S affects the physiology of both arrested and growing cells (12, 27).

All four of the GASP mutations examined in this study also increase the ability of the cell to utilize certain amino acids singly as the sole source of carbon and energy. These changes were manifested as either a higher growth rate or a new ability to grow on the particular amino acid. The GASP mutations are pleiotropic in this respect, as each affects growth on several amino acids. The subsets of amino acids on which they confer a growth advantage are overlapping but distinct. In general, these subsets contain amino acids that enter catabolism along the same major degradative pathways (reviewed in reference 18) (Fig. 6). The sgaA allele confers a growth advantage on amino acids that enter the central metabolic pathway through aspartate and fumarate, while the sgaB and sgaC alleles confer growth advantages on amino acids that enter this pathway through pyruvate (the effect of sgaC on proline metabolism is the one exception). The rpoS819 mutation, on the other hand, affected both of these degradative pathways. It is tempting to speculate that the GASP mutations, especially rpoS819, alter the regulation of the enzymes of these degradative pathways. We are currently investigating the mechanistic bases of these physiological changes.

View larger version (21K): [in this window] [in a new window]   FIG. 6.   The primary catabolic pathways for the amino acids on which the GASP mutants have a growth advantage (reviewed by McFall and Newman [18]). The arrows indicate enzymatic steps between metabolites. Boxed amino acids denote amino acids that can serve as sole sources of carbon and energy for at least one of the six strains tested. Listed under the boxed amino acids are the loci whose GASP alleles confer a growth advantage on the amino acid. An asterisk indicates the GASP allele that confers the novel ability to grow on the amino acid.

Based on our findings, we propose a model for a physiological basis of GASP (Fig. 7). We hypothesize that the primary selective force acting on carbon-starved cells is the ability to scavenge carbon sources released by the dying cells for the purposes of cell maintenance and growth. Cells unable to obtain a sufficient supply of carbon and energy can no longer maintain activities essential for viability, and they die. The fact that in every case studied the GASP fitness correlates directly with the capacity to catabolize amino acids leads us to propose that the most significant nutrients released are catabolizable amino acids. At the onset of starvation, the population is composed almost entirely of cells of the parental genotype that compete with equal fitness for carbon sources. Hence, the death during the first few days of starvation is stochastic, as all parental cells have an equal chance of scavenging the nutrients and surviving. However, the rare mutants within the population expressing the GASP phenotype outcompete their parents for the carbon resources because of their enhanced catabolic capabilities. These advantages provide the GASP cells with enough resources not only to survive but to grow and divide during starvation conditions. Once the GASP mutants grow to a significant cell density, they effectively decrease the amount of carbon available to the parental cells. These parental cells die as a result and release their nutrients into the medium. This model involves two positive feedback loops which can account for both the rapid growth of the GASP mutant and the rapid death of the parent. Supporting our model is the finding that if the G_II strain ZK1141 lacks the respiratory enzyme NADH dehydrogenase I, essential for the utilization of several amino acids catabolized by G_II (29), it loses the GASP phenotype versus the G_I parental strain, ZK819 (38).

View larger version (21K): [in this window] [in a new window]   FIG. 7.   A model for the physiological basis of the GASP phenotype.

Interestingly, we observed that while G_I mutants with a single G_II allele grow faster than G_I with certain amino acids as sole carbon sources, they grow slower with others. This result may seem inconsistent with our model for the physiological basis of GASP. However, growth on mixed amino acids may more accurately reflect the growth of the GASP mutants during starvation, since all amino acids are likely to be released at similar rates from the dying cells. We have demonstrated that all four GASP alleles confer an overall advantage when growing on mixed amino acids (Casamino Acids). Hence, our results suggest that when the GASP mutants are competing with their parents for the complex mixtures of nutrients released by the dying cells, their faster growth on some amino acids outweighs their slower growth on others.