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Genetics and Growth Regulation in Salmonella enterica
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Diarmaid Hughes)
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1052
Keyword [en]
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
Identifiers
URN: urn:nbn:se:uu:diva-235224ISBN: 978-91-554-9100-0 (print)OAI: oai:DiVA.org:uu-235224DiVA: diva2:759385
Public defence
2014-12-16, B21, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2014-11-24 Created: 2014-10-29 Last updated: 2015-02-03
List of papers
1. Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
Open this publication in new window or tab >>Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, e90486- p.Article in journal (Other academic) Published
Abstract [en]

Salmonella enterica grows extremely slowly when it depends on tufA499 (encoding the Gln125Arg mutant form of EF-Tu) to drive protein synthesis. We screened a plasmid library for multi-copy suppressors of the slow growth phenotype and identified spoT as a candidate. The spoT gene encodes a dual function enzyme with both ppGpp synthetase and hydrolase activities. When spoT was cloned behind an arabinose-inducible promoter the growth rate of the mutant strain increased in response to arabinose addition. We found that the slow-growing mutant strain had a relatively high basal level of ppGpp during exponential growth in rich medium. Overexpression of spoT significantly reduced this level of ppGpp suggesting that inappropriately high ppGpp levels might cause the slow growth rate associated with tufA499. We tested this hypothesis by inactivating relA (codes for RelA, a ribosome-associated ppGpp synthetase) in the mutant strain. This inactivation decreased the level of ppGpp in the mutant strain and increased its growth rate. Based on these data we propose that ribosomes depending on tufA499 for their supply of ternary complex (EF-Tu•GTP•aa-tRNA) experience amino acid starvation and that RelA on these starving ribosomes produces an excess of the alarmone ppGpp. This results in a suboptimal partitioning of transcription activity between genes important for fast growth in rich medium and genes important for growth in a poor medium. Accordingly, mutant bacteria growing in a rich medium act physiologically as though they were growing in a nutrient-poor environment. We propose that this generates a vicious circle and contributes to the extreme slow-growth phenotype associated with mutant EF-Tu. Reducing the level of ppGpp increases the growth rate of the mutant because it breaks this circle and reduces the wasteful misdirection of resources in the cell.

Keyword
tufA; ppGpp; RelA; Salmonella enterica; growth regulation
National Category
Microbiology
Research subject
Microbiology; Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-159663 (URN)10.1371/journal.pone.0090486 (DOI)000332396200210 ()
Note

Jessica M. Bergman and Disa L. Hammarlöf contributed equally to this work.

Available from: 2011-10-06 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
2. Turnover of mRNAs is an essential function of RNase E
Open this publication in new window or tab >>Turnover of mRNAs is an essential function of RNase E
(English)Manuscript (preprint) (Other academic)
Keyword
rpsA, vacB, RNaseR, RelBE, RNA turnover
National Category
Microbiology in the medical area
Research subject
Biology with specialization in Microbiology; Microbiology; Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-235199 (URN)
Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2015-02-03
3. Autoregulation of the tufB operon in Salmonella
Open this publication in new window or tab >>Autoregulation of the tufB operon in Salmonella
2016 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 100, no 6, 1004-1016 p.Article in journal (Refereed) Published
Abstract [en]

In Salmonella enterica and related species, translation elongation factor EF-Tu is encoded by two widely separated but near-identical genes, tufA and tufB. Two thirds of EF-Tu is expressed from tufA with the remaining one third coming from tufB. Inactivation of tufA is partly compensated by a doubling in the amount of EF-TuB but the mechanism of this up-regulation is unknown. By experimental evolution selecting for improved growth rate in a strain with an inactive tufA we selected six different noncoding or synonymous point mutations close to the tufB start codon. Based on these results we constructed a total of 161 different point mutations around the tufB start codon, as well as tufB 3'-truncations, and measured tufB expression using tufB-yfp transcriptional and translational fusions. The expression data support the presence of two competing stem-loop structures that can form in the 5'-end of the tufB mRNA. Formation of the 'closed' structure leads to Rho-dependent transcriptional termination of the tufB mRNA. We propose a model in which translational speed is used as a sensor for EF-Tu concentration and where the expression of tufB is post-transcriptionally regulated. This model describes for the first time how expression of the most abundant Salmonella protein is autoregulated.

Keyword
Salmonella enterica, tufA, tufB, EF-Tu, Rho, post-transcriptional regulation
National Category
Microbiology in the medical area
Research subject
Biology with specialization in Microbiology; Microbiology; Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-235218 (URN)10.1111/mmi.13364 (DOI)000379687100008 ()26934594 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2009.0251Swedish Research Council, 621-2012-2188; 521-2013-2904
Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2017-12-05Bibliographically approved
4. Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies
Open this publication in new window or tab >>Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

Keyword
aceBAK, ackA-pta, acs, pka, growth in stationary phase, Salmonella Typhimurium
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-234281 (URN)10.1371/journal.pone.0109255 (DOI)000342591500086 ()25275605 (PubMedID)
Available from: 2014-10-15 Created: 2014-10-15 Last updated: 2017-12-05Bibliographically approved

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