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Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Diarmaid Hughes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, United States of America. (Diarmaid Hughes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Diarmaid Hughes)
2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 10, e109255- p.Article in journal (Refereed) Published
Abstract [en]

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 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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 10, e109255- p.
Keyword [en]
aceBAK, ackA-pta, acs, pka, growth in stationary phase, Salmonella Typhimurium
National Category
Microbiology in the medical area
Research subject
URN: urn:nbn:se:uu:diva-234281DOI: 10.1371/journal.pone.0109255ISI: 000342591500086PubMedID: 25275605OAI: diva2:755949
Available from: 2014-10-15 Created: 2014-10-15 Last updated: 2015-02-03Bibliographically approved
In thesis
1. Genetics and Growth Regulation in Salmonella enterica
Open this publication in new window or tab >>Genetics and Growth Regulation in Salmonella enterica
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications.

Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response.

To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E.

Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA.

Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 59 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1052
tufA, tufB, EF-Tu, ppGpp, Stringent response, RNase E, RNA turnover, Post-transcriptional regulation, rpoB, rpoS, Growth in stationary phase
National Category
Microbiology in the medical area
Research subject
Biology with specialization in Microbiology; Microbiology; Molecular Genetics; Biology with specialization in Molecular Evolution
urn:nbn:se:uu:diva-235224 (URN)978-91-554-9100-0 (ISBN)
Public defence
2014-12-16, B21, BMC, Husargatan 3, Uppsala, 09:00 (English)
Available from: 2014-11-24 Created: 2014-10-29 Last updated: 2015-02-03

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