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Autoregulation of the tufB operon in Salmonella
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. (Diarmaid Hughes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Diarmaid Hughes)
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.

Place, publisher, year, edition, pages
2016. Vol. 100, no 6, 1004-1016 p.
Keyword [en]
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: urn:nbn:se:uu:diva-235218DOI: 10.1111/mmi.13364ISI: 000379687100008PubMedID: 26934594OAI: oai:DiVA.org:uu-235218DiVA: diva2:759347
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
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1052
Keyword
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: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)
Opponent
Supervisors
Available from: 2014-11-24 Created: 2014-10-29 Last updated: 2015-02-03
2. Biased Evolution: Causes and Consequences
Open this publication in new window or tab >>Biased Evolution: Causes and Consequences
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In evolution alternative genetic trajectories can potentially lead to similar phenotypic outcomes. However, certain trajectories are preferred over others. These preferences bias the genomes of living organisms and the underlying processes can be observed in ongoing evolution.

We have studied a variety of biases that can be found in bacterial chromosomes and determined the selective causes and functional consequences for the cell. We have quantified codon usage bias in highly expressed genes and shown that it is selected to optimise translational speed. We further demonstrated that the resulting differences in decoding speed can be used to regulate gene expression, and that the use of ‘non-optimal’ codons can be detrimental to reading frame maintenance. Biased gene location on the chromosome favours recombination between genes within gene families and leads to co-evolution. We have shown that such recombinational events can protect these gene families from inactivation by mobile genetic elements, and that chromosome organization can be selectively maintained because inversions can lead to the formation of unstable hybrid operons.

We have used the development of antibiotic resistance to study how different bacterial lifestyles influence evolutionary trajectories. For this we used two distinct pairs of antibiotics and disease-causing bacteria, namely (i) Mycobacterium tuberculosis that is treated with rifampicin and (ii) Escherichia coli that is treated with ciprofloxacin. We have shown that in the slow-growing Mycobacterium tuberculosis, resistance mutations are selected for high-level resistance. Fitness is initially less important, and over time fitness costs can be ameliorated by compensatory mutations. The need for rapid growth causes the selection of ciprofloxacin resistance in Escherichia coli not only to be selected on the basis of high-level resistance but also on high fitness. Compensatory evolution is therefore not required and is not observed.

Taken together, our results show that the evolution of a phenotype is the product of multiple steps and that many factors influence which trajectory is the most likely to occur and be most beneficial. Over time, selection will favour this particular trajectory and lead to biased evolution, affecting genome sequence and organization.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1198
Keyword
Evolution, Codon usage bias, Post-transcriptional regulation, Recombination, Inversion, EF-Tu, Frameshift suppression, Antibiotic resistance, Rifampicin, Ciprofloxacin, Compensatory evolution, Drug efflux, RNA polymerase, DNA gyrase
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-276456 (URN)978-91-554-9518-3 (ISBN)
Public defence
2016-05-09, A1:107a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2016-04-13 Created: 2016-02-13 Last updated: 2016-04-21

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