Long-term Stationary Phase Research Articles

Escherichia coli adaptation under prolonged resource exhaustion is characterized by extreme parallelism and frequent historical contingency.

Like many other non-sporulating bacterial species, Escherichia coli is able to survive prolonged periods of resource exhaustion, by entering a state of growth called long-term stationary phase (LTSP). In July 2015, we initiated a set of evolutionary experiments aimed at characterizing the dynamics of E. coli adaptation under LTSP. In these experiments populations of E. coli were allowed to initially grow on fresh rich media, but where not provided with any new external growth resources since their establishment. Utilizing whole genome sequencing data obtained for hundreds of clones sampled at 12 time points spanning the first six years of these experiments, we reveal several novel aspects of the dynamics of adaptation. First, we show that E. coli continuously adapts genetically, up to six years under resource exhaustion, through the highly convergent accumulation of mutations. We further show that upon entry into LTSP, long-lasting lineages are established. This lineage structure is in itself convergent, with similar lineages arising across independently evolving populations. The high parallelism with which adaptations occur under LTSP, combined with the LTSP populations' lineage structure, enable us to screen for pairs of loci displaying a significant association in the occurrence of mutations, suggestive of a historical contingency. We find that such associations are highly frequent and that a third of convergently mutated loci are involved in at least one such association. Combined our results demonstrate that LTSP adaptation is characterized by remarkably high parallelism and frequent historical contingency.

Pseudomonas putida Dynamics of Adaptation under Prolonged Resource Exhaustion.

Many nonsporulating bacterial species survive prolonged resource exhaustion, by entering a state termed long-term stationary phase. Here, we performed long-term stationary phase evolutionary experiments on the bacterium Pseudomonas putida, followed by whole-genome sequencing of evolved clones. We show that P. putida is able to persist and adapt genetically under long-term stationary phase. We observed an accumulation of mutations within the evolving P. putida populations. Within each population, independently evolving lineages are established early on and persist throughout the 4-month-long experiment. Mutations accumulate in a highly convergent manner, with similar loci being mutated across independently evolving populations. Across populations, mutators emerge, that due to mutations within mismatch repair genes developed a much higher rate of mutation than other clones with which they coexisted within their respective populations. While these general dynamics of the adaptive process are quite similar to those we previously observed in the model bacterium Escherichia coli, the specific loci that are involved in adaptation only partially overlap between P. putida and E. coli.

Disruption of trehalose periplasmic recycling dysregulates cAMP-CRP signaling in Escherichia coli during stationary phase.

Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such reserves. How Escherichia coli manage carbon storage systems under starvation stress, as well as transpose changes in intracellular metabolite levels into regulatory signals, is not well understood. Endogenous trehalose metabolism may be at the center of these processes, coupling carbon storage with carbon starvation responses. The coupled transport to the periplasm and subsequent hydrolysis of trehalose back to glucose for transport to the cytoplasm may function as a crucial metabolic signaling pathway. Although trehalose has been characterized as a stress protectant in E. coli, the disaccharide also functions as both an energy storage compound and a regulator of carbohydrate metabolism in fungi, plants, and other bacteria. Our research explores the metabolic regulatory properties of trehalose in E. coli and a potential mechanism by which the intracellular carbon pool is interconnected with regulatory circuits, enabling long-term survival.

Glycogen availability and pH variation in a medium simulating vaginal fluid influence the growth of vaginal Lactobacillus species and Gardnerella vaginalis

BackgroundGlycogen metabolism by Lactobacillus spp. that dominate the healthy vaginal microbiome contributes to a low vaginal pH (3.5–4.5). During bacterial vaginosis (BV), strict and facultative anaerobes including Gardnerella vaginalis become predominant, leading to an increase in the vaginal pH (> 4.5). BV enhances the risk of obstetrical complications, acquisition of sexually transmitted infections, and cervical cancer. Factors critical for the maintenance of the healthy vaginal microbiome or the transition to the BV microbiome are not well defined. Vaginal pH may affect glycogen metabolism by the vaginal microflora, thus influencing the shift in the vaginal microbiome.ResultsThe medium simulating vaginal fluid (MSVF) supported growth of L. jensenii 62G, L. gasseri 63 AM, and L. crispatus JV-V01, and G. vaginalis JCP8151A at specific initial pH conditions for 30 d. L. jensenii at all three starting pH levels (pH 4.0, 4.5, and 5.0), G. vaginalis at pH 4.5 and 5.0, and L. gasseri at pH 5.0 exhibited the long-term stationary phase when grown in MSVF. L. gasseri at pH 4.5 and L. crispatus at pH 5.0 displayed an extended lag phase over 30 d suggesting inefficient glycogen metabolism. Glycogen was essential for the growth of L. jensenii, L. crispatus, and G. vaginalis; only L. gasseri was able to survive in MSVF without glycogen, and only at pH 5.0, where it used glucose. All four species were able to survive for 15 d in MSVF with half the glycogen content but only at specific starting pH levels – pH 4.5 and 5.0 for L. jensenii, L. gasseri, and G. vaginalis and pH 5.0 for L. crispatus.ConclusionsThese results suggest that variations in the vaginal pH critically influence the colonization of the vaginal tract by lactobacilli and G. vaginalis JCP8151A by affecting their ability to metabolize glycogen. Further, we found that L. jensenii 62G is capable of glycogen metabolism over a broader pH range (4.0–5.0) while L. crispatus JV-V01 glycogen utilization is pH sensitive (only functional at pH 5.0). Finally, our results showed that G. vaginalis JCP8151A can colonize the vaginal tract for an extended period as long as the pH remains at 4.5 or above.

Metabolic adaptation to consume butyrate under prolonged resource exhaustion.

Bacteria must often survive following the exhaustion of their external growth resources. Fitting with this need, many bacterial species that cannot sporulate, can enter a state known as long term stationary phase (LTSP) in which they can persist for years within spent media. Several recent studies have revealed the dynamics of genetic adaptation of Escherichia coli under LTSP. Yet, the metabolic consequences of such genetic adaptation were not addressed. Here, we characterized the metabolic changes LTSP populations experience, over the first 32 days under LTSP. This allowed us to link genetic adaptations observed in a convergent manner across LTSP populations back to their metabolic adaptive effect. Specifically, we demonstrate that through the acquisition of mutations combinations in specific sets of metabolic genes, E. coli acquires the ability to consume the short chain fatty acid butyrate. Intriguingly, this fatty acid is not initially present within the rich media we used in this study. Instead, it is E. coli itself that produces butyrate during its initial growth within fresh rich media. The mutations that enable butyrate consumption allow E. coli to grow on butyrate. However, the clones carrying these mutations rapidly decrease in frequency, once the butyrate is consumed, likely reflecting an associated cost to fitness. Yet despite this, E. coli populations show a remarkable capability of maintaining these genotypes at low frequency, as standing variation. This in turn allows them to more rapidly re-adapt to consume butyrate, once it again becomes available to them.

Inference of transcriptome signatures of Escherichia coli in long-term stationary phase

“Non-growing” is a dominant life form of microorganisms in nature, where available nutrients and resources are limited. In laboratory culture systems, Escherichia coli can survive for years under starvation, denoted as long-term stationary phase, where a small fraction of cells manages to survive by recycling resources released from nonviable cells. Although the physiology by which viable cells in long-term stationary phase adapt to prolonged starvation is of great interest, their genome-wide response has not been fully understood. In this study, we analyzed transcriptional profiles of cells exposed to the supernatant of 30-day long-term stationary phase culture and found that their transcriptome profiles displayed several similar responses to those of cells in the 16-h short-term stationary phase. Nevertheless, our results revealed that cells in long-term stationary phase supernatant exhibit higher expressions of stress-response genes such as phage shock proteins (psp), and lower expressions of growth-related genes such as ribosomal proteins than those in the short-term stationary phase. We confirmed that the mutant lacking the psp operon showed lower survival and growth rate in the long-term stationary phase culture. This study identified transcriptional responses for stress-resistant physiology in the long-term stationary phase environment.

Cancer Stem Cells and Their Drug Resistance Mechanisms

Today, the prevalence rate of cancer continues to rise, and the mortality rate remains the first. In the face of tumor growth and deterioration, medical treatment methods are constantly updated, and drug resistance in the later stage of tumor is a thorny problem facing today. Cancer stem cells (CSCs) are an important factor leading to tumor drug resistance. The biological characteristics and drug resistance of tumor cells are important factors that determine tumor drug resistance. CSCs have unique biological characteristics, but they are similar to stem cells. It can self-proliferate and differentiate, have strong repair ability, can promote infinite growth of tumor, and have unique biological markers. The ABC protein drug pump function of CSCs can cope with the invasion of chemotherapy drugs. Its own microenvironment, strong inhibition of apoptosis and long-term stationary phase are the unique protective mechanism of CSCs. Aiming at the mechanism of cancer stem cells, the research on their properties is particularly important.

Bacterial strategies in prolonged stationary phase: Tradeoff between cell growth, maintenance, and recycling

In continuous starvation, bacteria will enter an extended stationary phase after the death phase. Growth-advantaged genetic mutations gradually emerge in the long-term stationary phase. The different competition patterns between younger and aged cultures, and the constant density and heterogeneity of living cells in very-aged cultures indicate the complexity of population dynamics under prolonged starvation. Based on resource conservation and protein allocation, here we constructed a simple growth-death-recycling model to describe the population dynamics of one or two bacterial strains during long-term starvation. Through analytical derivation or numerical simulation, we produced the survival-extinction phase diagram of one strain at steady state, and that of two strains at steady state, in a limited detection time, or consuming different nutrients. By relating allocated proteomic fractions to growth, death, and recycling rates, we showed the effects of proteome allocation on the phase diagrams. The constraints and optimization of protein allocation lead to the tradeoff between processes for cell growth, maintenance (repair), and nutrient recycling. The results reveal multiple strategies of bacterial survival and coexistence during long-term starvation.

The role of sigma factor competition in bacterial adaptation under prolonged starvation.

The study of adaptive microbial evolution in the laboratory can illuminate the genetic mechanisms of gaining fitness under a pre-defined set of selection factors. Laboratory evolution of bacteria under long-term starvation has gained importance in recent years because of its ability to uncover adaptive strategies that overcome prolonged nutrient limitation, a condition often encountered by natural microbes. In this evolutionary paradigm, bacteria are maintained in an energy-restricted environment in a growth phase called long-term stationary phase (LTSP). This phase is characterized by a stable, viable population size and highly dynamic genetic changes. Multiple independent iterations of LTSP evolution experiments have given rise to mutants that are slow-growing compared to the ancestor. Although the antagonistic regulation between rapid growth and the stress response is well-known in bacteria (especially Escherichia coli), the growth deficit of many LTSP-adapted mutants has not been explored in detail. In this review, I pinpoint the trade-off between growth and stress response as a dominant driver of evolutionary strategies under prolonged starvation. Focusing on mainly E. coli-based research, I discuss the various affectors and regulators of the competition between sigma factors to occupy their targets on the genome, and assess its effect on growth advantage in stationary phase (GASP). Finally, I comment on some crucial issues that hinder the progress of the field, including identification of novel metabolites in nutrient-depleted media, and the importance of using multidisciplinary research to resolve them.

Vibrio harveyi Exhibits the Growth Advantage in Stationary Phase Phenotype during Long-Term Incubation.

ABSTRACTThe bioluminescent marine bacterium Vibrio harveyi can exist within a host, acting as a mutualist or a parasitic microbe, and as planktonic cells in open seawater. This study demonstrates the ability of V. harveyi populations to survive and adapt under nutrient stress conditions in the laboratory, starting in an initially rich medium. V. harveyi populations remain viable into long-term stationary phase, for at least 1 month, without the addition of nutrients. To determine whether these communities are dynamic, populations were sampled after 10, 20, and 30 days of incubation and examined for their competitive ability when cocultured with an unaged, parental population. While populations incubated for 10 or 20 days showed some fitness advantage over parental populations, only after 30 days of incubation did all populations examined outcompete parental populations in coculture, fully expressing the growth advantage in stationary phase (GASP) phenotype. The ability to express GASP, in the absence of additional nutrients after inoculation, verifies the dynamism of long-term stationary-phase V. harveyi populations, implies the ability to generate genetic diversity, and demonstrates the plasticity of the V. harveyi genome, allowing for rapid adaptation for survival in changing culture environments. Despite the dynamism, the adaptation to the changing culture environment occurs less rapidly than in Escherichia coli, possibly due to Vibrio harveyi’s lower mutation frequency.IMPORTANCE Vibrio harveyi populations exist in many different niches within the ocean environment, as free-living cells, symbionts with particular squid and fish species, and parasites to other marine organisms. It is important to understand V. harveyi’s ability to survive and evolve within each of these niches. This study focuses on V. harveyi’s lifestyle outside the host environment, demonstrating this microbe’s ability to survive long-term culturing after inoculation in an initially rich medium and revealing increased competitive fitness correlated with incubation time when aged V. harveyi populations are cocultured with unaged, parental cultures. Thus, this study highlights the development of the growth advantage in stationary phase (GASP) phenotype in V. harveyi populations suggesting a dynamic population with fluctuating genotype frequencies throughout long-term, host-independent incubation.

Effect of initial seeding density on cell behavior-driven epigenetic memory and preferential lineage differentiation of human iPSCs

Understanding the cellular behavioral mechanisms underlying memory formation and maintenance in human induced pluripotent stem cell (hiPSC) culture provides key strategies for achieving stability and robustness of cell differentiation. Here, we show that changes in cell behavior-driven epigenetic memory of hiPSC cultures alter their pluripotent state and subsequent differentiation. Interestingly, pluripotency-associated genes were activated during the entire cell growth phases along with increased active modifications and decreased repressive modifications. This memory effect can last several days in the long-term stationary phase and was sustained in the aspect of cell behavioral changes after subculture. Further, changes in growth-related cell behavior were found to induce nucleoskeletal reorganization and active versus repressive modifications, thereby enabling hiPSCs to change their differentiation potential. Overall, we discuss the cell behavior-driven epigenetic memory induced by the culture environment, and the effect of previous memory on cell lineage specification in the process of hiPSC differentiation.

Dynamicsof Adaptation During Three Years of Evolution Under Long-Term Stationary Phase.

Many bacterial species that cannot sporulate, such as the model bacterium Escherichia coli, can nevertheless survive for years, following exhaustion of external resources, in a state termed long-term stationary phase (LTSP). Here we describe the dynamics of E. coli adaptation during the first three years spent under LTSP. We show that during this time, E. coli continuously adapts genetically through the accumulation of mutations. For nonmutator clones, the majority of mutations accumulated appear to be adaptive under LTSP, reflected in an extremely convergent pattern of mutation accumulation. Despite the rapid and convergent manner in which populations adapt under LTSP, they continue to harbor extensive genetic variation. The dynamics of evolution of mutation rates under LTSP are particularly interesting. The emergence of mutators affects overall mutation accumulation rates as well as the mutational spectra and the ultimate spectrum of adaptive alleles acquired under LTSP. With time, mutators can evolve even higher mutation rates through the acquisition of additional mutation rate–enhancing mutations. Different mutator and nonmutator clones within a single population and time point can display extreme variation in their mutation rates, resulting in differences in both the dynamics of adaptation and their associated deleterious burdens. Despite these differences, clones that vary greatly in their mutation rates tend to coexist within their populations for many years, under LTSP.

Effect of Initial Seeding Density on Cell Behavior-Driven Epigenetic Memory and Preferential Lineage Differentiation of Human iPSCs

Understanding the cellular behavioral mechanisms underlying memory formation and maintenance in human induced pluripotent stem cell (hiPSC) culture provides key strategies for achieving stability and robustness of cell differentiation. Here, we show that changes in cell behavior-driven epigenetic memory of hiPSC cultures alter their pluripotent state and subsequent differentiation. Interestingly, pluripotency-associated genes were activated during the entire cell growth phase along with increased active modifications and decreased repressive modifications. This memory effect can last several days in the long-term stationary phase and is sustained in the aspect of cell behavioral changes after subculture. Further, changes in growth-related cell behavior were found to induce nucleoskeletal reorganization, thereby enabling hiPSCs to change their ratio of active and repressive histone modifications and their differentiation potential. Overall, we discuss the cell behavior-driven epigenetic memory induced by the culture environment, and the effect of previous memory on cell lineage specification in the process of hiPSC differentiation.

Culture Volume Influences the Dynamics of Adaptation under Long-Term Stationary Phase

Escherichia coli and many other bacterial species, which are incapable of sporulation, can nevertheless survive within resource exhausted media by entering a state termed long-term stationary phase (LTSP). We have previously shown that E.coli populations adapt genetically under LTSP in an extremely convergent manner. Here, we examine how the dynamics of LTSP genetic adaptation are influenced by varying a single parameter of the experiment-culture volume. We find that culture volume affects survival under LTSP, with viable counts decreasing as volumes increase. Across all volumes, mutations accumulate with time, and the majority of mutations accumulated demonstrate signals of being adaptive. However, positive selection appears to affect mutation accumulation more strongly at higher, compared with lower volumes. Finally, we find that several similar genes are likely involved in adaptation across volumes. However, the specific mutations within these genes that contribute to adaptation can vary in a consistent manner. Combined, our results demonstrate how varying a single parameter of an evolutionary experiment can substantially influence the dynamics of observed adaptation.

DNA cytosine methyltransferase enhances viability during prolonged stationary phase in Escherichia coli.

In Escherichia coli, DNA cytosine methyltransferase (Dcm) methylates the second cytosine in the sequence 5'CCWGG3' generating 5-methylcytosine. Dcm is not associated with a cognate restriction enzyme, suggesting Dcm impacts facets of bacterial physiology outside of restriction-modification systems. Other than gene expression changes, there are few phenotypes that have been identified in strains with natural or engineered Dcm loss, and thus Dcm function has remained an enigma. Herein, we demonstrate that Dcm does not impact bacterial growth under optimal and selected stress conditions. However, Dcm does impact viability in long-term stationary phase competition experiments. Dcm+ cells outcompete cells lacking dcm under different conditions. Dcm knockout cells have more RpoS-dependent HPII catalase activity than wild-type cells. Thus, the impact of Dcm on stationary phase may involve changes in RpoS activity. Overall, our data reveal a new role for Dcm during long-term stationary phase.

Adaptations Accumulated under Prolonged Resource Exhaustion Are Highly Transient.

Many nonsporulating bacterial species can survive for years within exhausted growth media in a state termed long-term stationary phase (LTSP). We have been carrying out evolutionary experiments aimed at elucidating the dynamics of genetic adaptation under LTSP. We showed that Escherichia coli adapts to prolonged resource exhaustion through the highly convergent acquisition of mutations. In the most striking example of such convergent adaptation, we observed that across all independently evolving LTSP populations, over 90% of E. coli cells carry mutations to one of three specific sites of the RNA polymerase core enzyme (RNAPC). These LTSP adaptations reduce the ability of the cells carrying them to grow once fresh resources are again provided. Here, we examine how LTSP populations recover from costs associated with their adaptation once resources are again provided to them. We demonstrate that due to the ability of LTSP populations to maintain high levels of standing genetic variation during adaptation, costly adaptations are very rapidly purged from the population once they are provided with fresh resources. We further demonstrate that recovery from costs acquired during adaptation under LTSP occurs more rapidly than would be possible if LTSP adaptations had fixed during the time populations spent under resource exhaustion. Finally, we previously reported that under LTSP, some clones develop a mutator phenotype, greatly increasing their mutation accumulation rates. Here, we show that the mechanisms by which populations recover from costs associated with fixed adaptations may depend on mutator status.IMPORTANCE Many bacterial species can survive for decades under starvation, following the exhaustion of external growth resources. We have previously shown that bacteria genetically adapt under these conditions in a manner that reduces their ability to grow once resources again become available. Here, we study how populations that have been subject to very prolonged resource exhaustion recover from costs associated with their adaptation. We demonstrate that rapid adaptations acquired under prolonged starvation tend to be highly transient, rapidly reducing in frequency once bacteria are no longer starved. Our results shed light on the longer-term consequences of bacterial survival under prolonged starvation. More generally, these results may also be applicable to understanding longer-term consequences of rapid adaptation to additional conditions as well.

Escherichia coli Has a Unique Transcriptional Program in Long-Term Stationary Phase Allowing Identification of Genes Important for Survival.

Microbes live in complex and constantly changing environments, but it is difficult to replicate this in the laboratory. Escherichia coli has been used as a model organism in experimental evolution studies for years; specifically, we and others have used it to study evolution in complex environments by incubating the cells into long-term stationary phase (LTSP) in rich media. In LTSP, cells experience a variety of stresses and changing conditions. While we have hypothesized that this experimental system is more similar to natural environments than some other lab conditions, we do not yet know how cells respond to this environment biochemically or physiologically. In this study, we began to unravel the cells' responses to this environment by characterizing the transcriptome of cells during LTSP. We found that cells in LTSP have a unique transcriptional program and that several genes are uniquely upregulated or downregulated in this phase. Further, we identified two genes, cspB and cspI, which are most highly expressed in LTSP, even though these genes are primarily known to respond to cold shock. By competing cells lacking these genes with wild-type cells, we show that these genes are also important for survival during LTSP. These data can help identify gene products that may play a role in survival in this complex environment and lead to identification of novel functions of proteins.IMPORTANCE Experimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses that cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study, we began to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more-natural conditions.

Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability.

Purine metabolism plays a ubiquitous role in the physiology of Mycobacterium tuberculosis and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for M. tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Mycobacterium smegmatis Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in M. smegmatis but is not required for in vitro growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of M. tuberculosis HGPRT inhibitors displayed an unexpected antimicrobial activity against M. smegmatis that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed.IMPORTANCE Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5'-monophosphate from guanine and inosine-5'-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene (Δhgprt) in the model organism Mycobacterium smegmatis confirmed that this enzyme is not essential for M. smegmatis growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from Mycobacterium tuberculosis, displayed anti-M. smegmatis activities comparable to those obtained for M. tuberculosis but also inhibited the ΔhgprtM. smegmatis strain. These results confirmed that ANPs act in M. smegmatis by a mechanism independent of HGPRT.

Nanocalorimetry Reveals the Growth Dynamics of Escherichia coli Cells Undergoing Adaptive Evolution during Long-Term Stationary Phase.

Bacterial populations in long-term stationary-phase (LTSP) laboratory cultures can provide insights into physiological and genetic adaptations to low-energy conditions and population dynamics in natural environments. While overall population density remains stable, these communities are very dynamic and are characterized by the rapid emergence and succession of distinct mutants expressing the growth advantage in stationary phase (GASP) phenotype, which can reflect an increased capacity to withstand energy limitations and environmental stress. Here, we characterize the metabolic heat signatures and growth dynamics of GASP mutants within an evolving population using isothermal calorimetry. We aged Escherichia coli in anaerobic batch cultures over 20 days inside an isothermal nanocalorimeter and observed distinct heat events related to the emergence of three mutant populations expressing the GASP phenotype after 1.5, 3, and 7 days. Given the heat produced by each population, the maximum number of GASP mutant cells was calculated, revealing abundances of ∼2.5 × 107, ∼7.5 × 106, and ∼9.9 × 106 cells in the populations, respectively. These data indicate that mutants capable of expressing the GASP phenotype can be acquired during the exponential growth phase and subsequently expressed in LTSP culture.IMPORTANCE The present study is innovative in that we have identified previously unknown growth dynamics related to the temporal expression of the growth advantage in stationary phase (GASP) phenotype that allow mutants in long-term stationary-phase cultures to capitalize on the decrease of energy over prolonged incubation periods. By remaining in an active, but growth-limited, metabolic state similar to that observed in GASP cells grown in vitro, natural microbial communities might be able to prevail over much longer time scales. We believe this report to be a remarkable methodological and conceptual breakthrough in the study of the long-term survival and evolution of bacteria.

Survival and inactivation of Salmonella enterica serovar Typhimurium on food contact surfaces during log, stationary and long-term stationary phases

One of the most important transmission routes of foodborne pathogens is through contaminated food contact surfaces. In this study, we investigated the survival and inactivation of Salmonella enterica serovar Typhimurium on frequently used food contact surfaces in household settings, including plastic cutting board (CB), formica laminate (LA), and stainless steel (SS) surfaces. S. Typhimurium at log (6 h), stationary (24 h), or long-term stationary (LTS) (14 days) phases were evaluated. Results showed that, at medium and high microbial loads, LTS phase cells had significantly higher survivor population compared to log and stationary phase cells at 24 h after inoculation (p < 0.05). Disinfection study showed that LTS phase cells were very resilient to sodium hypochlorite when the initial microbial load was high with disinfection efficacy ranging from 26 to 35%. Scanning electron microscopy of these surfaces at 24 h after inoculation with a high microbial load revealed an early biofilm structure.