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Macromolecular Matchmaking: Mechanisms and Biology of Bacterial Small RNAs
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability.

By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , p. 66
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 916
Keyword [en]
Small RNA, sRNA, non-coding RNA, antisense, gene regulation, post-transcriptional regulation, translational control, outer membrane protein, OmpA, MicA, biofilm, flagella, curli, bistability, Lrp, MicF, CsgD, FlgM, OmrA, OmrB
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-171642ISBN: 978-91-554-8331-9 (print)OAI: oai:DiVA.org:uu-171642DiVA: diva2:511972
Public defence
2012-05-16, Sal B42, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-04-24 Created: 2012-03-25 Last updated: 2012-08-01Bibliographically approved
List of papers
1.
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2. Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis
Open this publication in new window or tab >>Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis
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2010 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 11, p. 1840-1850Article in journal (Refereed) Published
Abstract [en]

Escherichia coli produces proteinaceous surface structures called curli that are involved in adhesion and biofilm formation. CsgD is the transcriptional activator of curli genes. We show here that csgD expression is, in part, controlled post-transcriptionally by two redundant small RNAs (sRNAs), OmrA and OmrB. Their overexpression results in curli deficiency, in accordance with the inhibition of chromosomally encoded, FLAG-tagged CsgD. Downregulation of csgD occurs by a direct antisense interaction within the csgD 5'-UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmid-borne csgD-gfp fusions in vivo, and inhibit CsgD translation in vitro. The RNA chaperone Hfq is required for normal csgD mRNA and OmrA/B levels in the cell, and enhances sRNA-dependent inhibition of csgD translation in vitro. Translational inhibition involves two phylogenetically conserved secondary structure modules that are supported by chemical and enzymatic probing. The 5'-most element is necessary and sufficient for regulation, the one downstream comprises the RBS and affects translational efficiency. OmrA/B are two antisense RNAs that regulate a transcription factor to alter a morphotype and group behaviour.

Keyword
antisense RNA, bacterial sRNA, curli regulation, Hfq, transcriptional activator
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-131164 (URN)10.1038/emboj.2010.73 (DOI)000278235100007 ()20407422 (PubMedID)
Available from: 2010-09-26 Created: 2010-09-26 Last updated: 2017-12-12Bibliographically approved
3. A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp
Open this publication in new window or tab >>A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp
2012 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 84, no 3, p. 414-427Article in journal (Refereed) Published
Abstract [en]

Roughly 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, which responds to nutrient availability. Bioinformatically, we identified lrp as one of several putative targets for the sRNA MicF, which is transcriptionally downregulated by Lrp. Deleting micF results in higher Lrp levels, while overexpression of MicF inhibits Lrp synthesis. This effect is by antisense; mutations in the predicted interaction region relieve MicF-dependent repression of Lrp synthesis, and regulation is restored by compensatory mutations. In vitro, MicF sterically interferes with initiation complex formation and inhibits lrp mRNA translation. In vivo, MicF indirectly activates genes in the Lrp regulon by repressing Lrp, and causes severely impaired growth in minimal medium, a phenotype characteristic of lrp deletion strains. The double negative feedback between MicF and Lrp may promote a switch for adequate Lrp-dependent adaptation to nutrient availability. Lrp adds to the growing list of transcription factors that are targeted by sRNAs, thus indicating that perhaps the majority of all bacterial genes may be directly or indirectly controlled by sRNAs.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2012
Keyword
MicF, Lrp, sRNA, transcription factor, feedback regulation
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-171639 (URN)10.1111/j.1365-2958.2012.07994.x (DOI)000303050900003 ()22324810 (PubMedID)
Available from: 2012-03-25 Created: 2012-03-25 Last updated: 2017-12-07Bibliographically approved
4. Regulation of bacterial motility and bistable gene expression by base-pairing sRNAs
Open this publication in new window or tab >>Regulation of bacterial motility and bistable gene expression by base-pairing sRNAs
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-171636 (URN)
Available from: 2012-03-25 Created: 2012-03-25 Last updated: 2014-05-28

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