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Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies.

ABSTRACT: When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase ?-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.

SUBMITTER: Bergman JM

PROVIDER: S-EPMC4183559 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies.

Bergman Jessica M JM Wrande Marie M Hughes Diarmaid D

PloS one 20141002 10

When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase β-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several ...[more]

PMID: 25275605

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Project description:IntroductionThe American Heart Association (AHA) recommends regionalized care following out-of-hospital cardiac arrest (OHCA) at cardiac resuscitation centers (CRCs). Key level 1 CRC criteria include 24/7 percutaneous coronary intervention (PCI) capability, therapeutic hypothermia capability, and annual volume of ≥40 patients resuscitated from OHCA. Our objective was to characterize the availability and utilization of resources relevant to post-cardiac arrest care, including level 1 CRCs in California.MethodsWe combined data from the AHA, the California Office of Statewide Health Planning and Development (OSHPD), and surveys to identify CRCs. We surveyed emergency department directors and nurse managers at all 24/7 PCI centers identified by the AHA to determine their post-OHCA care capabilities. The survey included questions regarding therapeutic hypothermia use and specialist availability and was pilot-tested prior to distribution. Cases of OHCA were identified in the 2011 OSHPD Patient Discharge Database using a "present on admission" diagnosis of cardiac arrest (ICD-9-CM code 427.5). We defined key level 1 CRC criteria as 24/7 PCI capability, therapeutic hypothermia, and annual volume ≥40 patients admitted with a "present on admission" diagnosis of cardiac arrest. Our primary outcome was the proportion of hospitals meeting these criteria. Descriptive statistics and 95% CI are presented.ResultsOf the 333 acute care hospitals in California, 31 (9.3%, 95% CI 6.4-13%) met level 1 CRC criteria. These hospitals treated 25% (1937/7780; 95% CI 24-26%) of all admitted OHCA patients in California in 2011. Of the 125 hospitals identified as 24/7 PCI centers by the AHA, 54 (43%, 95% CI 34-52%) admitted ≥40 patients following OHCA in 2011. Seventy (56%, 95% CI 47-65%) responded to the survey; 69/70 (99%, 95% CI 92-100%) reported having a therapeutic hypothermia protocol in effect by 2011. Five percent of admitted OHCA patients (402/7780; 95% CI 4.7-5.7%) received therapeutic hypothermia and 18% (1372/7780; 95% CI 17-19%) underwent cardiac catheterization.ConclusionApproximately 10% of hospitals met key criteria for AHA level 1 CRCs. These hospitals treated one-quarter of patients resuscitated from OHCA in 2011. The feasibility of regionalized care for OHCA requires detailed evaluation prior to widespread implementation.

Project description:Hypermutable loci are widespread in bacteria as mechanisms for rapid generation of phenotypic diversity within a population that enables survival of fluctuating, often antagonistic, selection pressures. Localized hypermutation can mediate phase variation and enable survival of bacteriophage predation due to high frequency, reversible alterations in the expression of phage receptors. As phase variation can also generate population-to-population heterogeneity, we hypothesized that this phenomenon may facilitate survival of spatially-separated bacterial populations from phage invasion in a manner analogous to herd immunity to infectious diseases in human populations. The lic2A gene of Haemophilus influenzae is subject to "ON" and "OFF" switches in expression mediated by mutations in a 5'CAAT repeat tract present within the reading frame. The enzyme encoded by lic2A mediates addition of a galactose moiety of the lipopolysaccharide. This moiety is required for attachment of the HP1C1 phage such that the ON state of the lic2A gene is associated with HP1c1 susceptibility while the OFF state is resistant to infection. We developed an "oscillating prey assay" to examine phage spread through a series of sub-populations of Haemophilus influenzae whose phage receptor is in an ON or OFF state. Phage extinction was frequently observed when the proportion of phage-resistant sub-populations exceeded 34%. In silico modeling indicated that phage extinction was interdependent on phage loss during transfer between sub-populations and the frequency of resistant sub-populations. In a fixed-area oscillating prey assay, heterogeneity in phage resistance was observed to generate vast differences in phage densities across a meta-population of multiple bacterial sub-populations resulting in protective quarantining of some sub-populations from phage attack. We conclude that phase-variable hypermutable loci produce bacterial "herd immunity" with resistant intermediary-populations acting as a barricade to reduce the viral load faced by phage-susceptible sub-populations. This paradigm of meta-population protection is applicable to evolution of hypermutable loci in multiple bacteria-phage and host-pathogen interactions.

Project description:BackgroundIn this study, we aimed at investigating heterogeneity in the expression of metabolic genes in clonal populations of Escherichia coli growing on glucose as the sole carbon source. Different metabolic phenotypes can arise in these clonal populations through variation in the expression of glucose transporters and metabolic enzymes. First, we focused on the glucose transporters PtsG and MglBAC to analyze the diversity of glucose uptake strategies. Second, we analyzed phenotypic variation in the expression of genes involved in gluconeogenesis and acetate scavenging (as acetate is formed and excreted during bacterial growth on glucose), which can reveal, for instance, phenotypic subpopulations that cross-feed through the exchange of acetate. In these experiments, E. coli MG1655 strains containing different transcriptional GFP reporters were grown in chemostats and reporter expression was measured with flow cytometry.ResultsOur results suggest heterogeneous expression of metabolic genes in bacterial clonal populations grown in glucose environments. The two glucose transport systems exhibited different level of heterogeneity. The majority of the bacterial cells expressed the reporters for both glucose transporters MglBAC and PtsG and a small fraction of cells only expressed the reporter for Mgl. At a low dilution rate, signals from transcriptional reporters for acetyl-CoA synthetase Acs and phosphoenolpyruvate carboxykinase Pck indicated that almost all cells expressed the genes that are part of acetate utilization and the gluconeogenesis pathway, respectively. Possible co-existence of two phenotypic subpopulations differing in acs expression occurred at the threshold of the switch to overflow metabolism. The overflow metabolism results in the production of acetate and has been previously reported to occur at intermediate dilution rates in chemostats with high concentration of glucose in the feed.ConclusionsAnalysis of the heterogeneous expression of reporters for genes involved in glucose and acetate metabolism raises new question whether different metabolic phenotypes are expressed in clonal populations growing in continuous cultures fed on glucose as the initially sole carbon source.

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Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies.

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Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies.

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