A Mutant RNA Polymerase Activates the General Stress Response, Enabling Escherichia coli Adaptation to Late Prolonged Stationary Phase.

Escherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after 3 weeks of maintaining an E. coli K-12 population in stationary phase. This variant displayed a small colony phenotype and slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC(A494V) mutation confers the slow growth and small colony phenotype on this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary-phase sigma factor σS The RpoC(A494V) mutation upregulates the σS regulon. As shown over 20 years ago, early in prolonged stationary phase, σS attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σS activity, either by mutating the gene for σS directly or via mutations such as RpoC(A494V). The balance between the activities of the housekeeping major sigma factor and σS sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase.IMPORTANCE An important general mechanism of a bacterium's adaptation to its environment involves adjusting the balance between growing fast and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favor of the general stress response about 2 weeks later. This can be established by direct mutations in the master regulator of the general stress response or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance although the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

Using stochastic cell division and death to probe minimal units of cellular replication.

The invariant cell initiation mass measured in bacterial growth experiments has been interpreted as a minimal unit of cellular replication. Here we argue that the existence of such minimal units induces a coupling between the rates of stochastic cell division and death. To probe this coupling we tracked live and dead cells in Escherichia coli populations treated with a ribosome-targeting antibiotic. We find that the growth exponent from macroscopic cell growth or decay measurements can be represented as the difference of microscopic first-order cell division and death rates. The boundary between cell growth and decay, at which the number of live cells remains constant over time, occurs at the minimal inhibitory concentration (MIC) of the antibiotic. This state appears macroscopically static but is microscopically dynamic: division and death rates exactly cancel at MIC but each is remarkably high, reaching 60% of the antibiotic-free division rate. A stochastic model of cells as collections of minimal replicating units we term 'widgets' reproduces both steady-state and transient features of our experiments. Sub-cellular fluctuations of widget numbers stochastically drive each new daughter cell to one of two alternate fates, division or death. First-order division or death rates emerge as eigenvalues of a stationary Markov process, and can be expressed in terms of the widget's molecular properties. High division and death rates at MIC arise due to low mean and high relative fluctuations of widget number. Isolating cells at the threshold of irreversible death might allow molecular characterization of this minimal replication unit.

Genomewide Mutational Diversity in Escherichia coli Population Evolving in Prolonged Stationary Phase.

Prolonged stationary phase is an approximation of natural environments presenting a range of stresses. Survival in prolonged stationary phase requires alternative metabolic pathways for survival. This study describes the repertoire of mutations accumulating in starving Escherichia coli populations in lysogeny broth. A wide range of mutations accumulates over the course of 1 month in stationary phase. Single nucleotide polymorphisms (SNPs) constitute 64% of all mutations. A majority of these mutations are nonsynonymous and are located at conserved loci. There is an increase in genetic diversity in the evolving populations over time. Computer simulations of evolution in stationary phase suggest that the maximum frequency of mutations observed in our experimental populations cannot be explained by neutral drift. Moreover, there is frequent genetic parallelism across populations, suggesting that these mutations are under positive selection. Finally, functional analysis of mutations suggests that regulatory mutations are frequent targets of selection. IMPORTANCE Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E. coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28 days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection.

Cellular Immune Responses to Live Attenuated Japanese Encephalitis (JE) Vaccine SA14-14-2 in Adults in a JE/Dengue Co-Endemic Area.

BACKGROUND: Japanese encephalitis (JE) virus (JEV) causes severe epidemic encephalitis across Asia, for which the live attenuated vaccine SA14-14-2 is being used increasingly. JEV is a flavivirus, and is closely related to dengue virus (DENV), which is co-endemic in many parts of Asia, with clinically relevant interactions. There is no information on the human T cell response to SA14-14-2, or whether responses to SA14-14-2 cross-react with DENV. We used live attenuated JE vaccine SA14-14-2 as a model for studying T cell responses to JEV infection in adults, and to determine whether these T cell responses are cross-reactive with DENV, and other flaviviruses. METHODS: We conducted a single arm, open label clinical trial (registration: clinicaltrials.gov NCT01656200) to study T cell responses to SA14-14-2 in adults in South India, an area endemic for JE and dengue. RESULTS: Ten out of 16 (62.5%) participants seroconverted to JEV SA14-14-2, and geometric mean neutralising antibody (NAb) titre was 18.5. Proliferation responses were commonly present before vaccination in the absence of NAb, indicating a likely high degree of previous flavivirus exposure. Thirteen of 15 (87%) participants made T cell interferon-gamma (IFNγ) responses against JEV proteins. In four subjects tested, at least some T cell epitopes mapped cross-reacted with DENV and other flaviviruses. CONCLUSIONS: JEV SA14-14-2 was more immunogenic for T cell IFNγ than for NAb in adults in this JE/DENV co-endemic area. The proliferation positive, NAb negative combination may represent a new marker of long term immunity/exposure to JE. T cell responses can cross-react between JE vaccine and DENV in a co-endemic area, illustrating a need for greater knowledge on such responses to inform the development of next-generation vaccines effective against both diseases. TRIAL REGISTRATION: clinicaltrials.gov (NCT01656200).

Human T cell responses to Japanese encephalitis virus in health and disease.

Japanese encephalitis (JE) virus (JEV) is an important cause of encephalitis in children of South and Southeast Asia. However, the majority of individuals exposed to JEV only develop mild symptoms associated with long-lasting adaptive immunity. The related flavivirus dengue virus (DENV) cocirculates in many JEV-endemic areas, and clinical data suggest cross-protection between DENV and JEV. To address the role of T cell responses in protection against JEV, we conducted the first full-breadth analysis of the human memory T cell response using a synthetic peptide library. Ex vivo interferon-γ (IFN-γ) responses to JEV in healthy JEV-exposed donors were mostly CD8(+) and targeted nonstructural (NS) proteins, whereas IFN-γ responses in recovered JE patients were mostly CD4(+) and targeted structural proteins and the secreted protein NS1. Among patients, a high quality, polyfunctional CD4(+) T cell response was associated with complete recovery from JE. T cell responses from healthy donors showed a high degree of cross-reactivity to DENV that was less apparent in recovered JE patients despite equal exposure. These data reveal divergent functional CD4(+) and CD8(+) T cell responses linked to different clinical outcomes of JEV infection, associated with distinct targeting and broad flavivirus cross-reactivity including epitopes from DENV, West Nile, and Zika virus.

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.