Mutation of a LysR-type regulator of antifungal activity results in a growth advantage in stationary phase phenotype in Pseudomonas aureofaciens PA147-2

The growth advantage in stationary phase (GASP) phenotype was shown to be present in two mutants lacking the antifungal phenotype (Af(-) mutants) of Pseudomonas aureofaciens PA147-2. Complementation demonstrated a correlation between GASP and the antifungal defect in one strain but not in the second. Sequence analysis revealed the Af(-) GASP strain had a mutation in a gene (finR) encoding a LysR-type regulator. Antifungal-minus mutants arose in starved cultures, and those aged cultures had increased fitness. Taken together, the results show that there are at least two paths to the GASP phenotype in P. aureofaciens, one of which results in a concomitant loss of the antifungal phenotype.     

Colony dimorphism associated with stress resistance in Oenococcus oeni VP01 cells during stationary growth phase

The resistance to stresses as starvation, the presence of ethanol, sulfite and low pH, is a fundamental prerequisite for starter cultures used to induce malolactic fermentation in wine. In order to evaluate stress resistance of cells undergone starvation, cells viability in laboratory cultures of Oenococcus oeni VP01 strain was monitored during prolonged stationary growth phase. Once entered the stationary phase, strain VP01 showed 99% reduction of cell viability within 4 days. The remaining cells population maintained viability over 70 days and, when plated on agar medium, generated small colonies. The occurrence of this phenomenon was associated to stress resistance, since 10-day-old cells resulted more resistant than 3-day-old cells to ethanol and low pH conditions. No genomic mutations were revealed by pulse-field gel electrophoresis (PFGE) analysis in aged cultures. Total protein analysis by bidimensional electrophoresis highlighted differential protein expression in cultures differentially aged. It was demonstrated that O. oeni starving cultures at the stationary phase are constituted by dynamic cell populations. These results offer interesting perspective for a better understanding of cells behavior when inoculated in wine.     

Mutations Enhancing Amino Acid Catabolism Confer a Growth Advantage in Stationary Phase

Starved cultures of Escherichia coli undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the rpoS819 allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). Here we have identified three new GASP loci from an aged rpoS819 strain: sgaA, sgaB, and sgaC. Each locus is capable of conferring GASP on the rpoS819 parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells.     

A DNA polymerase V homologue encoded by TOL plasmid pWW0 confers evolutionary fitness on Pseudomonas putida under conditions of environmental stress

Plasmids in conjunction with other mobile elements such as transposons are major players in the genetic adaptation of bacteria in response to changes in environment. Here we show that a large catabolic TOL plasmid, pWW0, from Pseudomonas putida carries genes (rulAB genes) encoding an error-prone DNA polymerase Pol V homologue which increase the survival of bacteria under conditions of accumulation of DNA damage. A study of population dynamics in stationary phase revealed that the presence of pWW0-derived rulAB genes in the bacterial genome allows the expression of a strong growth advantage in stationary phase (GASP) phenotype of P. putida. When rulAB-carrying cells from an 8-day-old culture were mixed with Pol V-negative cells from a 1-day-old culture, cells derived from the aged culture out-competed cells from the nonaged culture and overtook the whole culture. At the same time, bacteria from an aged culture lacking the rulAB genes were only partially able to out-compete cells from a fresh overnight culture of the parental P. putida strain. Thus, in addition to conferring resistance to DNA damage, the plasmid-encoded Pol V genes significantly increase the evolutionary fitness of bacteria during prolonged nutritional starvation of a P. putida population. The results of our study indicate that RecA is involved in the control of expression of the pWW0-encoded Pol V.     

Mutation of a LysR-Type Regulator of Antifungal Activity Results in a Growth Advantage in Stationary Phase Phenotype in Pseudomonas aureofaciens PA147-2

The growth advantage in stationary phase (GASP) phenotype was shown to be present in two mutants lacking the antifungal phenotype (Af⁻ mutants) of Pseudomonas aureofaciens PA147-2. Complementation demonstrated a correlation between GASP and the antifungal defect in one strain but not in the second. Sequence analysis revealed the Af⁻ GASP strain had a mutation in a gene (finR) encoding a LysR-type regulator. Antifungal-minus mutants arose in starved cultures, and those aged cultures had increased fitness. Taken together, the results show that there are at least two paths to the GASP phenotype in P. aureofaciens, one of which results in a concomitant loss of the antifungal phenotype.     

Viability of escherichia coli cells under long-term cultivation in a rich nutrient medium

We investigated the viability of Escherichia coli cells during long-term cultivation in Brain Heart Infusion (BHI) medium and observed that the number of viable cells increased, then decreased, and increased again, in this medium, and finally the cells died out within about 10 days. This cell death may result from an increase in the pH of the medium. After repeated cultivation in BHI, bacterial cells that did not die out even under conditions of further cultivation were obtainable from cultures showing a stabilized viable count. We propose that long-term cultivation in BHI medium is a good system for studying growth phase-specific events in E. coli cells, because the total life-cycle of a population of E. coli, including exponential growth, stationary phase, and extinction, can be seen during a period of only about 10 days. Also, this system clearly allows detection of a phenotype that may not be detectable in other commonly used media. Moreover, in this report, we show that mutants displaying the GASP (growth advantage in stationary phase) phenotype appear at high frequency under long-term cultivation conditions.     

Long-term survival during stationary phase: evolution and the GASP phenotype

The traditional view of the stationary phase of the bacterial life cycle, obtained using standard laboratory culture practices, although useful, might not always provide us with the complete picture. Here, the traditional three phases of the bacterial life cycle are expanded to include two additional phases: death phase and long-term stationary phase. In many natural environments, bacteria probably exist in conditions more akin to those of long-term stationary-phase cultures, in which the expression of a wide variety of stress-response genes and alternative metabolic pathways is essential for survival. Furthermore, stressful environments can result in selection for mutants that express the growth advantage in stationary phase (GASP) phenotype.     

Tat transport of linker-containing proteins in Escherichia coli

Little is known about the dynamics of cellular growth, death, and evolution within bacterial biofilms. Here we show evidence of evolution within single-species biofilms in real time. Escherichia coli harvested from 22-day-old biofilms express a competitive advantage over cells incubated in biofilms for shorter periods of time. This advantage is manifested as the ability of aged cells to outcompete younger cells in the presence of a pre-existing biofilm, even though cells from older biofilms do not express an increased ability to form initial biofilms on a fresh, unoccupied surface. This phenomenon is similar to the growth advantage in stationary phase, or GASP, phenotype exhibited by planktonically grown cells when incubated under competitive conditions. The ability of bacteria in biofilms to show rapid heritable change has implications for our understanding of the adaptive abilities of biofilms in a wide variety of natural and man-made environments.     

Involvement of the Global Regulator H-NS in the Survival of Escherichia coli in Stationary Phase

Long-term batch cultures of Escherichia coli grown in nutrient-rich medium accumulate mutations that provide a growth advantage in the stationary phase (GASP). We have examined the survivors of prolonged stationary phase to identify loci involved in conferring a growth advantage and show that a mutation in the hns gene causing reduced activity of the global regulator H-NS confers a GASP phenotype under specific conditions. The hns-66 allele bears a point mutation within the termination codon of the H-NS open reading frame, resulting in a longer protein that is partially functional. Although isolated from a long-term stationary-phase culture of the parent carrying the rpoS819 allele that results in reduced RpoS activity, the hns-66 survivor showed a growth disadvantage in the early stationary phase (24 to 48 h) when competed against the parent. The hns-66 mutant is also unstable and reverts at a high frequency in the early stationary phase by accumulating second-site suppressor mutations within the ssrA gene involved in targeting aberrant proteins for proteolysis. The mutant was more stable and showed a moderate growth advantage in combination with the rpoS819 allele when competed against a 21-day-old parent. These studies show that H-NS is a target for mutations conferring fitness gain that depends on the genetic background as well as on the stage of the stationary phase.     

Escherichia coli genes involved in cell survival during dormancy: role of oxidative stress.

When Escherichia coli cells reach stationary phase of growth, specific gene products are synthesized that protect cells while dormant. "Aged" cells may remain viable in cultures for years. For example, agar cultures stored for 38 years still had more than 10(5) viable cells/ml. However, when specific mutants were cultured, the population of these mutants dropped sharply after 4-10 days. This defect is termed "Stationary-Phase-Death". Each mutant strain was hypersensitive to near-ultraviolet radiation and other oxidative agents. Bovine catalase rescued many of the mutants from death in dormancy, suggesting that specific gene products protect "aged" cells against oxidative damage.     

The nature of laboratory domestication changes in freshly isolated Escherichia coli strains

Adaptation of environmental bacteria to laboratory conditions can lead to modification of important traits, what we term domestication. Little is known about the rapidity and reproducibility of domestication changes, the uniformity of these changes within a species or how diverse these are in a single culture. Here, we analysed phenotypic changes in nutrient‐rich liquid media or on agar of four Escherichia coli strains newly isolated through minimal steps from different sources. The laboratory‐cultured populations showed changes in metabolism, morphotype, fitness and in some phenotypes associated with the sigma factor RpoS. Domestication events and phenotypic diversity started to emerge within 2–3 days in replicate subcultures of the same ancestor. In some strains, increased amino acid usage and higher fitness under nutrient limitation resembled those in mutants with the GASP (growth advantage in stationary phase) phenotype. The domestication changes are not uniform across a species or even within a single domesticated population. However, some parallelism in adaptation within repeat cultures was observed. Differences in the laboratory environment also determine domestication effects, which differ between liquid and solid media or with extended stationary phase. Important lessons for the handling and storage of organisms can be based on these studies.     

GASP phenotype: presence in enterobacteria and independence of sigmaS in its acquisition

The appearance of growth advantage in stationary phase or GASP was originally detected in Escherichia coli. The presence of this phenotype in other enterobacteria such as Enterobacter cloacae, Salmonella typhimurium, Providencia stuartii and Shigella dysenteriae is described in this work. E. cloacae GASP strains presented lower levels of RpoS than the parental strain, although no mutation in the gene or its promoter was detected. This work offers evidence of GASP rpoS-independent pathways as GASP was also acquired in knock-out rpoS E. cloacae and E. coli strains.     

Response of Escherichia coli to ethyl methanesulfonate: Influence of growth phase and repair ability on survival and mutagenesis

The effects of altering the cell growth rate (physiological state) and DNA repair capacity (genetic state) on susceptibility to inactivation and mutagenesis by ethyl methanesulfonate (EMS) were studied in 4 strains of E. coli. Logarithmic and stationary phase cells of the polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇ recA, and the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃ rec⁺, were exposed to EMS and the surviving fraction and mutant frequency determined. At the same EMS concentration both mutants were more susceptible to inactivation than the parental strains. In all 4 strains, log phase cells were more sensitive to inactivation than stationary cells. The surviving fraction of stationary cells exceeded log cells by a factor of 18 for polA, 6 for recA, and about 2 for the parental strains. In all strains, except recA, log phase cells exhibited higher spontaneous mutant frequencies than stationary phase cells. At the same concentration of EMS, survivors of both polA and recA showed more than 10-fold higher induced frequencies than the wild types. However, at the same survival levels the repair deficient mutants exhibited induced mutant frequencies comparable to the repair proficient strains. There was no significant effect of growth phase on EMS induced mutability in recA or the parental strains. In marked contrast, the polymerase I deficient mutant shows both a higher spontaneous frequency and a greater than 10-fold higher EMS induced mutant frequency in log phase cultures compared to stationary phase cultures. Our results support the hypothesis that cellular susceptibility to alkylating agents is influenced by both the genetic capability for repair and the particular physiological state of the cell.     

Anomalous spatial redistribution of competing bacteria under starvation conditions

Bacterial cells evolved under prolonged stress often have a growth advantage in stationary phase (GASP); we expect GASP cells to maintain a proliferative state and dominate wild-type cells during starvation, especially when nutrients are limited and the medium has been conditioned. However, when we compete GASP mutants against wild-type cells in a chain of microfluidic microhabitat patches (MHPs) with alternating nutrient-rich and nutrient-limited regions, we observe the reverse effect: wild-type cells achieve maximum relative density under nutrient-limited conditions, while GASP cells dominate nutrient-rich regions. We explain this surprising observation in terms of ideal free distributions, where we show that wild-type cells maximize their fitness at high cell density by redistributing themselves to sparsely populated MHPs. At the microscopic level, we describe how biofilm formation also contributes to the population redistribution. We conclude by discussing the implications of these results for social interactions of more complex organisms.     

微生物生活周期中的一个新的时期——长期稳定期

传统的对微生物生长时期的认识一般分为4个时期:迟缓期、对数期、稳定期、死亡期。实际上,这种划分不足以使我们认识到微生物生长过程的全貌。近年来许多研究表明,在死亡期之后存在一个完全不同意义的时期——长期稳定期。这个时期可能与微生物在环境中的生存状态更加相似。微生物细胞通过突变得以生存,并在选择中形成稳定期生长优势表型,深入研究微生物长期稳定期具有极其重要的意义。     

Morphological adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus

During exponential growth, each cell cycle of the α-purple bacterium Caulobacter crescentus gives rise to two different cell types: a motile swarmer cell and a sessile stalked cell. When cultures of C. crescentus are grown for extended periods in complex (PYE) medium, cells undergo dramatic morphological changes and display increased resistance to stress. After cultures enter stationary phase, most cells are arrested at the predivisional stage. For the first 6–8 days after inoculation, the colony-forming units (cfu) steadily decrease from 10⁹ cfu ml⁻¹ to a minimum of 3 × 10⁷ cfu ml⁻¹ after which cells gradually adopt an elongated helical morphology. For days 9–12, the cfu of the culture increase and stabilize around 2 × 10⁸ cfu ml⁻¹. The viable cells have an elongated helical morphology with no constrictions and an average length of 20 μm, which is 15–20 times longer than exponentially growing cells. The level of the cell division initiation protein FtsZ decreases during the first week in stationary phase and remains at a low constant level consistent with the lack of cell division. When resuspended in fresh medium, the elongated cells return to normal size and morphology within 12 h. Cells that have returned from stationary phase proceed through the same developmental changes when they are again grown for an extended period and have not acquired a heritable growth advantage in stationary phase (GASP) compared with overnight cultures. We conclude that the changes observed in prolonged cultures are the result of entry into a new developmental pathway and are not due to mutation.     

Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12

Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(S) (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an rpoS strain had acquired a GASP mutation in lrp. This GASP allele, lrp-1141, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The lrp-1141 allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a lrp null allele generated by a transposon insertion has a fitness gain identical to that of the lrp-1141 allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the lrp-1141 GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the lrp GASP phenotype, and hence the lrp GASP phenotype is due to more global physiological changes.     

Lipid Cytochemistry and Morphologic Change in Aging Populations of Tetrahymena

SYNOPSIS. In cultures of strains GL, S, W, and WH6 of Tetrahymena pyriformis grown in proteose-peptone medium and stained with phosphine 3R the percentage of cells containing lipid granules increased in early stationary phase, decreased in the middle of stationary phase, and increased again toward the end of culture life. Granules appeared first in the anterior ends of cells and spread thruout the cytoplasm as the cultures aged. These granules consisted of neutral fat and were present in living cells. Changes in lipid accumulation were correlated with changes in cellular size, shape, volume and fragility.