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Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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.

Place, publisher, year, edition, pages
2014. Vol. 9, no 2, e90486- p.
Keyword [en]
tufA; ppGpp; RelA; Salmonella enterica; growth regulation
National Category
Microbiology
Research subject
Microbiology; Molecular Cellbiology
Identifiers
URN: urn:nbn:se:uu:diva-159663DOI: 10.1371/journal.pone.0090486ISI: 000332396200210OAI: oai:DiVA.org:uu-159663DiVA: diva2:446168
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
In thesis
1. EF-Tu and RNase E: Essential and Functionally Connected Proteins
Open this publication in new window or tab >>EF-Tu and RNase E: Essential and Functionally Connected Proteins
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme.

We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode.

RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 863
Keyword
bacterial growth, translation, EF-Tu, RNase E, mRNA, RNA degradation
National Category
Microbiology
Research subject
Molecular Cellbiology; Microbiology
Identifiers
urn:nbn:se:uu:diva-159682 (URN)978-91-554-8179-7 (ISBN)
Public defence
2011-11-24, B21, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2011-11-03 Created: 2011-10-06 Last updated: 2011-11-10Bibliographically approved
2. 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

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