Escherichia coli evolution during stationary phase

Research in Microbiology 155 (2004) 328–336 www.elsevier.com/locate/resmic

Escherichia coli evolution during stationary phase Erik R. Zinser a , Roberto Kolter b,∗ a Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 400 Main St., Cambridge, MA 02139, USA b Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA

Received 7 October 2003; accepted 20 January 2004 Available online 19 March 2004

Abstract The process of evolution by natural selection has been known for a century and a half, yet the mechanics of selection are still poorly understood. In most cases where natural selection has been studied, the genetic and physiological bases of fitness variation that result in population changes were not identified, leaving only a partial understanding of selection. Starved cultures of the bacterium Escherichia coli present a model system with which to address the genetic and physiological bases of natural selection. This is a model system that also reflects the prevalent state of bacteria in the natural world; due to intense competition for nutrients, microorganisms spend the majority of their lives under starvation conditions. Genetic analyses of a single survivor of starvation identified four adaptive mutations1 . Investigation of these mutations has revealed insights into the molecular and physiological bases of evolution during prolonged starvation stress.  2004 Elsevier SAS. All rights reserved. Keywords: Escherichia coli; Natural selection; GASP; Adaptive mutations; Starvation stress

1. Background 1.1. Models of microbial evolution Three complementary laboratory systems exist that model the natural states of microbial populations. Serially-transferred batch cultures model evolution during a (semi) continuous growth of total population under nutrient-replete conditions (nutrient supply is in excess of demand, allowing for maximal growth rate) [8]. Chemostat cultures model evolution during nutrient-limited continuous growth (nutrient supply allows net population growth, but is less than the amount required for maximal growth rate) [6]. In contrast to the first two systems, the starved batch culture models evolution during periods of net population stasis or decline, whereby nutrient supply cannot sustain net population growth [11,41]. Though examples exist of natural microbial populations experiencing sustained growth (balanced by loss via predation or washout), starvation is thought to be * Corresponding author.

E-mail address: [email protected] (R. Kolter). 1 For the purposes of this review the term “adaptive mutation” refers to

mutations that are beneficial to organisms under the conditions tested. 0923-2508/$ – see front matter  2004 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2004.01.014

the prevalent state of most populations for the majority of their existence [21,22], and is the focus of this review. 1.2. The GASP phenomenon The starvation model for microbial evolution consists of batch cultures grown to stationary phase and incubated without further introduction of nutrients or cells. The majority of cells lose viability in the first few days of stationary phase, but then the rate of overall loss of viability rapidly declines [11]. Cultures of starved Escherichia coli retain viable counts even after five years of incubation [Finkel and Kolter, unpublished]. Under these conditions, cells with a growth advantage in stationary phase (GASP) phenotype arise by mutation, and grow within the culture to either coexist with the parental majority or displace the parent [40]. Evolution during stationary phase is a continuous process, as the populations are repeatedly taken over by mutants of increased fitness throughout the stationary phase period [10,40]. Mixed culture competitions between the evolved strain and the parent are used to demonstrate the GASP phenotype (i.e., the fitness gain) of the evolved strain. The GASP phenotype is due to adaptation resulting from selection, and is not merely the ability of aged cultures to outcompete unaged cultures due to physiological acclimation to starvation.

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Genetic crosses between GASP strains and their wild-type parents have demonstrated that the GASP phenotype is due to stable mutations [40,42]. The GASP phenotype was initially observed in starved E. coli grown aerobically in LB broth. Since then, the GASP phenotype has been observed in E. coli cultures grown in minimal glucose medium and in anaerobic LB broth [33,38], and in cultures of other bacterial species, including Shigella dysenteriae, Pseudomonas putida, and Mycobacterium smegmatis [7,9,29]. In all the cases reported above, except for P. putida, the cultures were starved for carbon; for P. putida, the cultures were starved for phosphate. Interestingly, the first report of population dynamics in stationary phase was for Serratia marcescens and Sarcina lutea in 1939 [31]. In this report the investigators noted significant fluctuations in population size in cultures starved for thirteen months, and suggested that there may be selection processes acting on the starved population. Evidence was also provided that the source of nutrition for these starvation survivors was the dying majority cells. Further evidence for this scavenging phenomenon is provided in Section 4 of this paper. Hence, starved microbial cultures represent a model system in which to study natural selection. The evolutionary process is continuous, and initial reports suggest that it is a generalizable phenomenon, and can occur for any microbial species exposed to any condition of prolonged starvation. 1.3. Genes that play positive roles in stationary phase fitness Two genetic approaches have been used to study the nature of selection in stationary phase. One approach is to mutate the strain in question (wild type or GASP mutants) and screen for mutations that reduce fitness. This approach provides insight into important physiological processes that are under selective pressure to be maintained (purifying selection). The second approach provides insight into processes under selective pressure to be changed (diversifying selection). Insights gained from the former approach are described presently, while the latter approach is the subject of the remainder of this review. Analysis of mutants with decreased fitness during stationary phase has (1) ascribed novel roles to known proteins, (2) identified the “missing” genes whose activities were previously known, and most interestingly, (3) identified novel activities never before detected in E. coli. Examples of these three types of findings are: 1. The SOS-induced DNA polymerases II, III, and V [37], the L-isoaspartyl protein repair methyltransferase [34], and the phage shock protein [36] have all been previously characterized, but have only recently been recognized to have novel roles in stationary phase fitness. 2. The electrogenic NADH dehydrogenase I enzyme of E. coli had been known for decades, yet the genetic

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locus coding for the enzyme remained elusive. This locus was identified in a screen for insertion mutations that disrupted the GASP phenotype of strain ZK1141 [39]. Hence, energy production via respiration was implicated as an important activity during growth and takeover by this mutant. 3. Another genetic screen for mutants of reduced stationary phase fitness identified a gene homologous to competence genes from Haemophilus influenzae and Neisseria gonorrhoeae [12]. Further investigations led to the discovery that E. coli is able to take up DNA and use it as a carbon and energy source (the role of competence in genetic transformation remains speculative for E. coli). 1.4. Outline of review There have been numerous experimental investigations of microbial evolution, but few have identified the genes involved in selection, especially multiple adaptive mutations found within an evolved clone. We believed that a more detailed molecular and physiological analysis could reveal new insights into the process of evolution by natural selection. In the sections that follow, we describe our recent characterization of a single GASP mutant. First, we describe the identities of three of the four GASP mutations found within this strain. Second, we discuss the molecular natures of the mutations themselves, and what they reveal about mutation and selection during stationary phase. Third, we show how the identification of adaptive mutations leads to testable hypotheses about the forces of selection during stationary phase. Finally, we discuss the broader evolutionary implications of certain discoveries made as a result of our detailed analyses of the beneficial mutations.

2. Identification of three GASP mutations in a single strain By nature the stationary phase evolutionary model restricts the growth of the majority population. Therefore, the progression of evolution over time cannot be measured in terms of generations. Instead, we have devised an arbitrary yet practical nomenclature to relate evolved mutants and their parents [42]. We define our laboratory wild-type strain (E. coli K-12 W3110) as G0 (GASP0 ). GI strains are isolated as survivors of starved G0 cultures that demonstrate a stationary phase fitness gain relative to their G0 parents. Likewise, GII strains are isolates from starved GI cultures that have a fitness gain relative to their GI parent, and so forth. Our discussion focuses on a single GI strain (ZK819) [40], and a single GII derivative of that strain (ZK1141) [39]. Genetic analyses confirmed that the GI GASP phenotype was due to a single mutation, whereas the GII GASP phenotype was due to three additional mutations found in different regions of the genome. The presence of all three

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GII mutations was determined to be both necessary and sufficient for the “complete” GII GASP phenotype of the aged survivor. In the following paragraphs we identify the GI GASP mutation and two of the three GII GASP mutations. The identity of the third GII mutation, sgaC, remains elusive.

2.1. The GI mutation is an rpoS allele Initial studies on the genetics of the GASP phenotype identified a partial loss-of-function allele of rpoS as the GI GASP mutations [40]. The rpoS locus encodes the stationary-phase sigma factor, σS , which is responsible for global changes in gene expression that take place at the onset of carbon starvation in E. coli [16]. σS -regulated processes change the physiological state of the cell in many ways: the overall metabolism of the cell is decreased, the resistances to starvation, pH, osmotic, temperature, and oxidative stresses are increased, and the cell’s length decreases. The nature of the rpoS allele in the GI GASP strain investigated (rpoS819) was a 46 base pair duplication resulting in a “reduced function” phenotype, i.e., all σS -dependent genes tested exhibited lower levels of expression [40]. The physiological basis of this fitness gain was unknown, but predicted to be due to pleiotropic effects (discussed below). 2.2. The GII GASP allele Lrp-1141 The lesion responsible for the GASP phenotype of one of the three GII mutations of this strain was mapped to the lrp gene, encoding the transcription factor, leucineresponsive regulatory protein (Lrp) [43]. Lrp, like σS , regulates many genes, and is induced during the transition into stationary phase [2,17,24,32]. The mutant GASP allele of lrp, Lrp-1141, was determined to be an in-frame threebase-pair deletion [43]. This deletion results in a protein product that lacks a glycine residue in the turn of the helix-turn-helix (HTH) DNA binding motif. Significantly, this glycine residue is almost universally-conserved among HTH proteins, and is thought to be important for the bend of the turn in the HTH motif [1,15]. Transcription studies of an Lrp-activated and an Lrp-repressed gene (gltB and sdaA, respectively) indicated that there was no residual activity in this protein [43]. These results are consistent with the mutant Lrp protein having a mis-folded DNA-binding domain that is unable to bind DNA and therefore preventing transcription regulation. An lrp null allele conferred the same GASP phenotype as the lrp allele from the starvation survivor, confirming that loss of Lrp activity results in a fitness gain in stationary phase. Despite the inability to bind DNA, however, the Lrp-1141 mutant protein appears stable and capable of forming inactive heteromultimers (dimers, octamers, and hexadecamers [2–4]) with wild-type protein, as indicated by analyses of merodiploid strains [43]. This dominant negative nature of Lrp-1141 is discussed below.

2.3. The GII GASP allele IN(cstA::IS5-IS5D) The lesion responsible for one of the other two GII GASP mutations was identified as a genomic rearrangement involving two IS5 insertion sequences [45]. Though the order of events that resulted in the final re-arrangement is unknown, it is easiest to describe the re-arrangement as a two-step mutation. In step one, an IS5 element transposed into the regulatory region of the starvation-inducible cstA gene, which encodes an oligopeptide permease [28]. In step two, an inversion event took place between this IS5 and a pre-existing IS5 about 60 kb away. This latter IS5 (IS5D) was positioned just upstream of a five-gene cluster, originally thought to compose the operon ybeJ–gltJKL– rihA. The first four genes (ybeJ–gltJKL) are predicted to encode a high affinity ABC-type transporter for aspartate and glutamate. The fifth gene (rihA, formerly ybeK) has been identified as a cytidine nucleoside hydrolase, and recent evidence suggests it is independent of the operon [25]. This insertion/inversion mutation, designated IN(cstA::IS5IS5D), simultaneously inactivated the cstA gene and activated the ybeJ–gltJKL operon [45]. The nature of this inactivation/activation phenomenon was determined to be due to the re-positioning of the CRP-binding region originally in the regulatory region of cstA to the upstream region of the ybeJ–gltJKL operon. This CRP Box sequence is responsible for the stationary phase-specific activation of cstA, and genetic analysis of transcription suggested that it was likewise playing a role in activating the ybeJ– gltJKL operon in the IN(cstA::IS5-IS5D) mutant in stationary phase.

3. Observations on the molecular nature of the GASP mutations Molecular characterization of the three known GASP mutations in the GII strain ZK1141 allows us to make some preliminary generalizations about the classes of mutations selected during evolution in stationary phase. Of course, greater sampling will be required to obtain higher confidence in these generalizations. Nevertheless, we provide them as a frame of reference for future studies of the system that will likely employ novel, high-throughput technologies to uncover GASP mutations at a faster rate than is possible with traditional genetics. 3.1. Selection favors a wide range of mutation types The molecular natures of the few known GASP mutations are surprisingly variable. The rpoS819 mutation of the GI strain was a short duplication [40]. In the GII strain we found two more types of GASP mutations: a small, in-frame deletion (lrp-1141) and a combination transposition/inversion (IN(cstA::IS5-IS5D)) involving mobile genetic elements (IS5’s) [43,45]. GASP alleles of rpoS in other

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GI strains were found to contain point mutations, including transitions, transversions, and a frameshift mutation [13,40]. It would appear that all classes of mutations can be adaptive in stationary phase. As each class will have its own inherent frequency, determination of the net rate of adaptive mutation must take each classes’ rate into account. 3.2. Selection favors altered gene expression rather than generation of novel function We found that the overall effect of the mutations tends to be an alteration in expression, rather than function. For the IN(cstA::IS5-IS5D) GASP mutant, we saw that the mutation increased the expression of the ybeJ–gltJKL operon, without any apparent change in the function of the protein products [45]. While the rpoS819 and Lrp-1141 mutations clearly altered the activity of the proteins that they encoded [40,43], the role of these proteins is in regulating gene expression. Hence, the physiological effects that they confer are also a result of altered gene expression, rather than the generation of novel gene function. The evolutionary implication of this finding is that adaptation to new environments is dominated, at least initially, by changes in the regulation of pre-existing gene activities, rather than the generation of new ones de novo. As we have seen, changes in gene expression can result from a single lesion, whereas significant changes in gene function may require multiple mutations (e.g., gene duplication followed by mutation(s) in the open reading frame). Exposure to prolonged starvation (for months, perhaps) may indeed select for altered function, and awaits confirmation in future studies. 3.3. Selection favors pleiotropic mutations Two of the three GASP genes identified thus far, rpoS and lrp, are global regulators of transcription in E. coli [2,16,24]. They coordinate metabolic and physiologic responses to environmental stresses by co-regulating genes of related function. Mutations in these regulators may therefore yield a higher net fitness increase than mutations that affect single loci. Studies in serially-transferred batch cultures of E. coli have similarly identified adaptive, pleiotropic mutations in spoT, which regulates the concentration of the effector molecule ppGpp [5]. It is noteworthy that clinical and soil isolates of E. coli and salmonellae have extensive allelic variation in rpoS, indicating that there is considerable selection pressure acting on global regulators in the natural environment [13,18,26,27,35]. While adaptive mutations are not found exclusively in global regulators, these results do suggest that pleiotropic mutations in general have a higher fitness potential than non-pleiotropic mutations, and may influence the relative contributions each type of mutation makes to evolutionary diversification [8,43].

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3.4. Does selection favor dominant mutations? The lrp GASP allele has a peculiar nature: when overexpressed it behaves as a dominant negative [43]. The Lrp1141 allele codes for a protein that has a single amino acid deletion in its DNA binding domain, making it unable to regulate transcription. However, this mutant protein is stable and can form inactive multimers when mixed with wildtype protein. It would seem improbable that such a dominant loss-of-function mutation was fortuitously found in a GASP survivor; if any loss-of-function allele can confer the GASP phenotype, then recessive mutations should occur at higher frequency in the population. Perhaps, however, the dominant nature of lrp-1141 was itself involved in the selective process. Dominant mutations have a time advantage over recessive ones, and this may be of crucial importance in stationary phase [43]. Recessive loss-of-function mutations have a significant phenotypic lag: due to the presence of wild-type products of the gene produced prior to mutation, the effects of mutation are not initially manifest in the cell. The cell remains phenotypically wild type until the wild-type protein is degraded or diluted by successive cell divisions. Under starvation conditions, the total population size is constant or in decay. Hence, dilution of wild-type protein by cell division is restricted, making proteolysis the determining factor in phenotypic lag time for recessive mutations. By comparison, dominant mutations can exert their effects immediately after their products are made, significantly reducing the phenotypic lag. Both types of mutations may occur within the population, but the dominant ones have a distinctly shorter phenotypic lag, which can give them a head start in population takeovers. We hypothesize that several factors determine the dominant or recessive nature of the GASP alleles present in the surviving cells. One is the mutation rate, which likely favors recessive mutations over dominant ones. Another factor is the phenotypic lag period of the adaptive mutations. This rate favors dominant mutations. Finally, there is the rate of stochastic death. Simply put, mutant cells may simply starve to death during a prolonged phenotypic lag due fitness-independent factors. This rate also favors dominant mutations. Of the three well-characterized GASP alleles, two of them are dominant: Lrp-1141 (dominant negative) and IN(cstA::IS5-IS5D) (gain-of-function) [43,45]. The nature of the rpoS819 GASP allele is unknown, but it is interesting to note that like the Lrp-1141 allele, the position of the mutation in rpoS819 is in the DNA-binding domain, not in the domain where it interacts with core RNA polymerase [40]. It is possible that it too can exhibit a dominantnegative effect by sequestering core RNA polymerase from wild type σS . Future analyses of GASP mutations should shed light on these important rates and evaluate the importance of phenotypic lag in the selection of adaptive mutations.

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4. Forces of selection revealed by the nature of the GASP mutations

4.2. GASP mutants grow faster on amino acids All four GASP mutations (including the unidentified GII mutation sgaC ) confer faster growth on mixtures of amino acids as the sole carbon source [42]. What is more, each GASP mutant outgrows its parent in media containing specific amino acids. Therefore, the growth characteristics of the GASP mutants were consistent with the hypothesis that GASP mutants grow by outcompeting their parents for amino acids in stationary phase.

4.1. The amino acid scavenging model for GASP One of the major goals for identifying the GASP mutations was to generate testable hypotheses about the forces of selection during starvation. The defining characteristic of GASP mutations is their ability to confer growth to minority populations under starvation conditions where the majority population size remains static or is in decay [40]. That these minority populations are able to grow indicates that starvation is not universally experienced amongst the total population. We predicted that nutrients are released as the majority of cells die, and selection favors mutants with enhanced abilities to scavenge these nutrients [42]. The rpoS regulon is quite diverse, consisting of genes involved in metabolism, but also cell morphology, resistance to oxidative, acid, base, and osmotic stress, to name a few [16]. This has made the a priori identification of the physiological basis of GASP by rpoS mutants difficult. However, identification of two of the GII GASP loci, lrp and ybeJ–gltJKL, provided evidence that amino acid scavenging is selected for during starvation [43,45]. Lrp is responsible for (among other things) activating amino acid anabolism while repressing amino acid catabolism [2,24]. Loss of Lrp function would result in a metabolic shift towards amino acid scavenging. Likewise, activation of the ybeJ– gltJKL operon in the GASP strain would increase the ability of the cell to scavenge aspartate and glutamate from the environment. Amino acids are the most abundant molecules of the cell, and can provide carbon, nitrogen, and sulfur to growing cells. Given such evidence, we hypothesized that starvation selects for enhanced amino acid scavenging and metabolism [42].

4.3. Faster growth on amino acids is a shared trait amongst different GII strains Examination of independent evolutionary lineages confirmed a strong selective pressure for enhancing amino acid scavenging in stationary phase [44]. Ten parallel cultures of the GI strain containing the rpoS819 GASP allele were aged for four weeks in LB broth. After four weeks, dilutions were grown on LB plates, and different colony morphotypes that arose were tested for a GII phenotype and an enhanced growth on amino acids, relative to their GI parent. In all ten cultures, GII GASP mutants were found, and almost all of them were able to grow faster than the GI parent on one or more amino acids (Table 1). Interestingly, almost all cultures had at least one morphotype that could outgrow its parent on aspartate and glutamate, suggesting a strong selective pressure exists for the ability to exploit these resources. The GI parent is a poor scavenger of these two amino acids, due to inefficient transport, and likely leaves these two resources relatively untouched in stationary phase [42]. Selection would therefore be strong for mutations like IN(cstA::IS5-IS5D) that increase transport activity and allow the cell to exploit these resources.

Table 1 Phenotypes of isolates from 10 parallel LB-grown cultures of the rpoS819 GI strain aged four weeks Culture 1 2 3 4 5 6 7 8 9 10

Morphotypes A, B A, B A, B A A, B, C, D A, B A, B A, B, C A, B, C A, B

Morphotypes expressing

Morphotypes with faster growth on:

a GII phenotype

SER

A, B A, B A, B A A, B, C A, B A, B A, B, C A, B, C A, B

A, B

A

THR

A C

C

PRO A A A A, B A B B

GLU

ASP

A, B A A, B A

A, B A A, B A

A A, B A, B, C

A A A, B A, B, C A, B

Isolates were chosen based on reproducible colony morphology differences (different morphotypes are designated arbitrarily with letters A, B, C, D. However, note that the morphotype letter designations refer to intra-culture differences, not inter-culture similarities between “A” morphotypes, and so on). The GII GASP phenotype of the morphotypes was determined as the ability to grow as a 1:1000 minority versus the GI parent. Relative growth rates compared to the GI parent were determined on solid (serine, proline, glutamate, and aspartate) or in liquid (threonine) minimal media. When multiple letters appear in a cell of the table it means that multiple morphotypes were observed.

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4.4. GASP mutants consume amino acids during growth Several lines of evidence suggest that the GASP mutants do indeed consume amino acids released by the dying majority. First, a wild-type culture grown in a defined medium lacking tryptophan was taken over by a GASP derivative that was unable to make its own tryptophan [42]. This event could not occur unless the GASP auxotroph satisfied its tryptophan requirement by scavenging from the prototrophic majority. A second line of evidence comes from an analysis of the medium composition during a GASP takeover event. We measured the amount of serine in the media with a serA bioassay strain [44]. This strain cannot grow without an exogenous supply of serine (though it can produce some serine from glycine [30]). Aliquots of the spent supernatants added to a minimal glucose medium constitute the sole source of serine for the serA strain to utilize, and growth yields are compared to yields on known quantities of serine. The advantage of the bioassay over chemical analyses is that it sums all bioavailable forms of serine, including serine found in mixed composition oligopeptides. In one bioassay experiment, a GI (rpoS819) strain consumed the serine component of LB during exponential growth (Fig. 1) [44]. Between days 1 and 4 of starvation, most of the population died, while the serine increased. The most likely source of the new serine is nutrients released by the dying cells. On day 5, the viable counts increased an order of magnitude, and the serine concentration dropped again. Analysis of colonies isolated on day 5 demonstrated that they were GII GASP mutants. Hence, as the GASP mutants took over the population, they consumed the serine released by the dying cells. It is important to note the logarithmic scales of the y-axes of Fig. 1. Most of the cells already died by day 2, but most of the serine did not become

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bioavailable until day 5. Either the cells did not release the serine until several days after they lost viability, or they released the serine when they died but the serine did not become bioavailable until a few days after. As K-12 strains of E. coli contain no known extracellular proteases, the decomposition of released proteins into bioavailable monomers and oligomers may be the rate-limiting step for the GASP takeovers. This decomposition of proteins into bioavailable sources may be augmented by the release of intracellular proteases into the medium during death and lysis of the majority. 4.5. Amino acid scavenging is not the whole story GASP mutants with the lrp-1141 allele grow faster on alanine, serine and threonine [42]. However, genetic constructs of the lrp-1141 mutant that are blocked in the pathways for the degradation of these amino acids still retain the GASP phenotype relative to the wild-type parent [43]. Therefore, the amino acid scavenging model is not sufficient to explain the fitness gain of the lrp-1141 mutant. However, the serine–glycine–threonine degradation pathway was shown to play a significant role in the GASP phenotype of the lrp mutants, confirming an important (but not essential) role of amino acid scavenging in conferring a stationary phase fitness gain [43]. Clearly, other physiological changes in the lrp-1141 mutant are as important or more important in conferring a fitness advantage. Whole genome microarray comparisons of wild type and lrp mutants provide suggestions for the physiological basis of GASP by lrp mutants [17,32]. Roughly 400 genes were found to be responsive to Lrp, including those involved in central intermediary metabolism and in the tolerance of pH and osmotic stress [32]. These newly identified members of the lrp regulon represent targets for future genetic analysis of the lrp GASP phenotype. Genetic analysis of the amino acid scavenging model of GASP has yet to be performed for the other known GASP mutations. As the IN(cstA::IS5-IS5D) mutation activates a transporter for glutamate and aspartate [45], it seems likely that the amino acid scavenging model is correct for this GASP mutation. However, like lrp, rpoS regulates many genes [16] and rpoS GASP mutants may likewise have a more complex basis of stationary phase fitness gain. Interestingly, a recent report demonstrates that E. coli consumes DNA during starvation, and mutants that cannot eat DNA suffer a significant fitness loss [12]. Perhaps in addition to amino acids, GASP mutants outcompete their parents for DNA released from the dying majority, contributing to an overall fitness gain.

5. Broader evolutionary implications Fig. 1. Serine bioassay of the supernatant of a starved GI culture. The rpoS819 GI strain was inoculated on day 0 into 50 ml LB, and aliquots of the culture were subjected to bioassay for serine.

As discussed in the introduction, patterns of evolution we observe in starved laboratory cultures should provide

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insight into the evolutionary processes extant in the natural world. In this section some of the broader implications of our molecular characterizations of the GASP mutations that were found in a single strain are discussed. 5.1. Evolution is rapid during prolonged starvation GASP-mediated takeovers are rapid in stationary phase. A culture of the GI strain aged only two weeks contained a survivor with three adaptive mutations [42]. In competition assays, secondary GASP mutations consistently arise in the majority and even the original minority population by the end of the experiment (usually 10 days) [44]. The rapid population turnover in starved cultures suggests evolution may be quite rapid in natural populations that experience long periods of starvation due to nutrient limitation. One of the factors contributing to the rapid rate of evolution is the nature of the environment. While it is homogeneous with respect to habitat (as a continuously-mixed culture), starved populations are flush with a wide range of potential nutrient resources generated by cell death and lysis of the majority [42,44]. While the total concentrations of these nutrients cannot sustain growth of the total population, they nevertheless provide great opportunity for the exploitation of new niches by mutants with altered physiology. 5.2. Evolution redefines the niche by shifts in resource utilization As stated previously, each GASP mutation of the GII strain enhances the cell’s ability to scavenge certain amino acids ([42] and Table 2). Significantly, with the exception of proline, these amino acids fall into one of two categories: those consumed faster by the IN(cstA::IS5-IS5D) mutant, and those consumed faster by the Lrp-1141 and the (unidentified) sgaC mutants (Table 2). Differential consumption of different resources may lead to the development of discrete niches in stationary phase, a theory tested by competition experiments. GI strains with one of each of the GII GASP mutations were competed head-to-head in reciprocal 1:1000 mixes [44]. The sgaC strain grew versus Lrp-1141, but not vice versa. The sgaC and Lrp-1141 mutations therefore enhance fitness in the same niche, consistent with their similar amino acid metabolism profiles. In contrast, the IN(cstA::IS5-IS5D) mutant grew versus sgaC

and lrp-1141, and vice versa. Therefore, the IN(cstA::IS5IS5D) mutation enhances fitness in a different niche than the other two, again consistent with their amino acid profiles. (cstA activity, which the IN(cstA::IS5-IS5D) mutant lacks, was not necessary for the sgaC and lrp-1141 strains to grow versus the IN(cstA::IS5-IS5D) mutant. This result indicates that their niche is not simply oligopeptides not consumed by the IN(cstA::IS5-IS5D) mutant (see below)). The presence of all three mutations in a single survivor indicates it has significantly expanded its metabolic capacity and likewise may have expanded its niche. Current studies are aimed at determining if this potential for niche expansion is realized during starvation, or if epistatic interactions between the adaptive mutations prevent it. Beneficial mutations can also act to reduce the metabolic capacity of the cell and diminish the niche. Inactivation of the cstA gene by the IN(cstA::IS5-IS5D) mutation led to a loss of a significant nutrient resource in stationary phase, namely oligopeptides [45]. Wild-type cells were able to exploit these resources when inoculated as a minority population into a IN(cstA::IS5-IS5D) culture, and were able to grow in stationary phase competitions [45]. This growth was confirmed to be due almost exclusively to CstA activity. The IN(cstA::IS5-IS5D) mutation thus represents a trade-off in resource scavenging ability: amino acid monomers versus oligopeptides. Analysis of the GII mutant demonstrates that adaptation can result in both expansion and reduction of the niche, resulting in a rapid and dramatic change of the physiological profile of the organism. 5.3. Loss of advantageous genes via selection As discussed above, the IN(cstA::IS5-IS5D) GASP mutation is a genomic rearrangement that results in changes in expression at two loci: a gain of expression of the ybeJ– gltJKL operon, and a loss of expression of the cstA locus [45]. Higher expression of the ybeJ–gltJKL operon allows a IN(cstA::IS5-IS5D) GASP mutant minority to invade a wild-type majority by enhancing the scavenging of amino acids. Significantly, cstA is also beneficial in stationary phase [45]. CstA is a stationary phase-induced oligopeptide permease [28], and cells expressing cstA can invade a majority population that cannot express it. Hence, the IN(cstA::IS5-IS5D) GASP mutant, as a consequence of the inversion mutation, has gained one beneficial activity only to lose another. In the IN(cstA::IS5-IS5D) mutation we see

Table 2 Growth phenotypes of the GII mutations GII GASP allele

IN(cstA::IS5-IS5D) lrp-1141 sgaC

Enhanced growth on:

Stationary phase growth as minority versus:

Ser

Thr

Ala

Pro

Glu

Asp

Asn

IN(cstA::IS5-IS5D)

lrp-1141

sgaC

− + +

− + +

− + +

+ − +

+ − −

+ − −

+ − −

− + +

+ − +

+ − −

Assays were performed in the rpoS819 GI background. Meaning of symbols: −, no enhancement of growth rate observed (left half of table) or no GASP phenotype observed (right half of table). +, enhancement of growth rate observed (left half of table) or GASP phenotype observed (right half of table).

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how selection can favor the loss of a beneficial gene activity. Expression of the ybeJ–gltJKL operon is beneficial, and is facilitated by the expropriation of the regulatory element of another beneficial gene. This provides a net fitness gain: by sacrificing its ability to compete for oligopeptides at equal fitness with its parent, it has gained the ability to outcompete its parent for monomeric amino acids. This loss of a beneficial gene activity is an example of a gene expression trade-off, and we speculate that this is a significant evolutionary mechanism. Horizontal gene transfer is thought to be the major mechanism of diversification in prokaryotes [14,19,20,23]. As noted by others, however, a fundamental problem exists with horizontal transfer: the transferred genes need to be expressed in order to be beneficial [19]. This problem may become more pronounced with greater evolutionary divergence between donor and host, as the upstream regulatory elements of the transferred genes need to be compatible with the transcription machinery of the host. Regulatory element expropriation, such as we have described for the IN(cstA::IS5-IS5D) mutation, could overcome this problem [45]. Simply, the horizontally-acquired genes could be expressed immediately if they are inserted downstream of a host gene’s promoter and/or regulatory element. Fitness losses due to host gene inactivation can be offset by the fitness gains resulting from expression of the foreign genes, especially if this allows for the exploitation of novel niches. Subsequent (permanent) loss of the inactivated gene of the host could then occur by mutation and drift, as mutations in an unexpressed gene are likely to be neutral.

lyze new GASP genes, in E. coli and other microbes, under our current conditions of starvation and under other starvation regimes (anaerobic starvation, starvation for nitrogen, etc.). In particular, we see great potential in the investigation of the interactions between successively acquired adaptive mutations. Do we see evidence for synergistic effects on fitness, or is interference possible? Are there physiological constraints that dictate the chronological appearance of adaptive mutations? Finally, how does adding complexity to the system, such as addition of phage or other microbes, affect the nature of adaptation during starvation?

6. Conclusions and future directions

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The objective of this review was to provide a framework for future investigations into the nature of microbial evolution during periods of prolonged starvation. As starvation is such a common state for microbes in nature, we believe there is much to be discovered about evolution using this model system. Using a molecular approach, we have already learned that evolution occurs very rapidly in starved populations, and takes advantage of different types of mutations. Selection appears to favor changes in gene expression over function, and may favor dominant mutations over recessive due to the influence of phenotypic lag. Starvation appears to select for mutants of E. coli with enhanced abilities to scavenge amino acids, although we know that the situation is more complex and may involve other metabolites as well, such as DNA. We have also seen that successive adaptive mutations shift the metabolic capacity of the cell and thus redefine its niche. Finally, a molecular analysis of the IN(cstA::IS5-IS5D) mutation provided evidence that adaptive mutations may involve the loss of beneficial genes. Future studies should either confirm or reject the hypotheses provided by these first glances into the forces of selection during starvation. It will be important to identify and ana-

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Acknowledgements We are grateful to Steve Finkel for critical reading of this manuscript. E.Z. is the recipient of a post-doctoral fellowship from the National Science Foundation. This review is dedicated to the memory of Michel Blot, outstanding colleague and good friend. I (R.K.) first met Michel during a Gordon Conference in 1993, when he presented his remarkable work on the movement of insertion sequences in different isolates of W3110 that had been kept in stabs. I have a vivid memory of his enthusiastic discussion of our own results in studying long-term cultures, something he termed “The Zambrano Effect” [40], the phenomenon now known as “GASP”. Since then, we had ongoing discussions and a collaboration whose published product [45] Michel sadly did not see. I think he would enjoy knowing that we are still “GASPing for Life in Stationary Phase”.

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