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Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Justus Liebig Univ, Inst Mikrobiol & Mol Biol, D-35392 Giessen, Germany..
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
2017 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, no 6, p. 1020-1033Article in journal (Refereed) Published
Abstract [en]

Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5 UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.

Place, publisher, year, edition, pages
WILEY , 2017. Vol. 103, no 6, p. 1020-1033
National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-320870DOI: 10.1111/mmi.13607ISI: 000395758900008PubMedID: 27997707OAI: oai:DiVA.org:uu-320870DiVA, id: diva2:1091180
Funder
German Research Foundation (DFG), BE 5210/1-1 BE 5210/2-1Swedish Research Council, VR 621-2010-5233Available from: 2017-04-26 Created: 2017-04-26 Last updated: 2018-02-28Bibliographically approved
In thesis
1. Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
Open this publication in new window or tab >>Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is nowadays widely accepted that non-coding RNAs play important roles in post-transcriptional regulation of genes in all kingdoms of life. In bacteria, the largest group of RNA regulators are the small RNAs (sRNAs). Almost all sRNAs act through anti-sense base-pairing with target mRNAs, and by doing so regulate their translation and/or stability. As important modulators of gene expression, sRNAs are involved in all aspects of bacterial physiology. My studies aimed to deepen our understanding of the mechanisms behind sRNA-mediated gene regulation. We have shown that translation of the di-guanylate-cyclase YdaM, a major player in the biofilm regulatory cascade, is repressed by the sRNAs OmrA and OmrB. OmrAB require the RNA chaperone protein Hfq for efficient regulation. Interestingly, our results suggest a non-canonical mechanism for Hfq-mediated ydaM-OmrA/B base-pairing. Instead of serving as RNA interaction platform, Hfq restructures the ydaM mRNA to enable sRNA binding. We also addressed the question of how bacteria utilize regulatory RNAs to create phenotypic heterogeneity by studying the role of the tisB/istR-1 type 1 toxin-antitoxin system in SOS-induced persister cell formation in E. coli.

In addition, I have investigated the prokaryotic CRISPR-Cas immune system, which has led to the development of two molecular tools. The CRISPR-Cas adaptive immune system consists of a CRISPR array, where palindromic repeats are interspaced by unique spacer sequences derived from foreign genetic elements, and the CRISPR-associated (Cas) proteins. In the adaptation stage, memory is created by insertion of spacer sequences into the CRISPR array. We developed a fluorescent reporter that accurately and sensitively detects spacer integration events (denoted: “acquisition”) in single cells and in real-time. In the effector stage of immunity, crRNAs, consisting of one spacer-repeat unit, associate with the Cas proteins to form a ribonucleoprotein complex that surveys the cell for invader DNA. Target identification occurs by base-pairing between the crRNA and the complementary sequence in the target nucleic acid, which triggers degradation. We repurposed the E. coli type I-E CRISPR-Cas effector complex Cascade for specific reprogrammable transcriptional gene silencing.

The studies presented herein thus contributes to our understanding of RNA-based target identification for gene regulation and adaptive immunity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 83
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1637
Keywords
small RNA, sRNA, non-coding RNA, Hfq, gene regulation, post-transcriptional regulation, biofilm, OmrA, OmrB, toxin-antitoxin, TisB, IstR-1, Persisters, CRISPR-Cas, CRISPR-Cas adaptation reporter, Escherichia coli
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-343526 (URN)978-91-513-0252-2 (ISBN)
Public defence
2018-04-20, A1:111a, BMC, Husargatan 3, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2018-03-26 Created: 2018-02-28 Last updated: 2018-04-24

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Berghoff, Bork A.Hoekzema, MirtheWagner, Gerhart E. H.

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