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The transcriptional regulator VanT activates expression of the signal synthase VanM forming a regulatory loop in the Vibrio anguillarum quorum sensing system
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Debra L. Milton)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Debra L. Milton)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Debra L. Milton)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In Vibrio anguillarum, one quorum-sensing regulatory sRNA, Qrr1, was previously shown to destabilize vanT mRNA repressing expression of the transcriptional regulator VanT. In this study, three additional Qrr sRNAs that destabilize vanT mRNA were identified. The expression of the Qrr sRNA depends on the active (phosphorylated) form of the σ54-dependent response regulator VanO and the sigma factor RpoN (σ54). The phosphotransferase VanU is predicted to activate VanO, but also a putative second response regulator that may repress qrr expression activating VanT expression. Signal production by the homoserine lactone synthase VanM inhibits the phosphorelay determining VanU activity. Therefore, qrr expression and VanT were analyzed in the ΔvanM mutant. Interestingly, VanM activated qrr expression and repressed VanT expression. Moreover, vanM expression is tightly regulated and peaks early in growth. Several putative Hfq binding sites are present in the vanM 5´-untranslated region. Thus, vanM expression and mRNA stability were measured in the Δhfq mutant. Hfq destabilizes vanM mRNA, but also regulates vanM transcription. The transcriptional effect occurs indirectly through VanT, which is derepressed in the Δhfq mutant. Additionally, VanT directly binds to a conserved binding motif in the vanM promoter to positively regulate vanM expression. In summary, VanM and VanT generate a regulatory loop, in which VanM induces qrr expression to repress VanT expression and VanT negatively regulates its own expression by activating vanM expression.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-33256OAI: oai:DiVA.org:umu-33256DiVA: diva2:311083
Available from: 2010-04-20 Created: 2010-04-20 Last updated: 2010-04-29Bibliographically approved
In thesis
1. Stress response and virulence in Vibrio anguillarum
Open this publication in new window or tab >>Stress response and virulence in Vibrio anguillarum
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacteria use quorum sensing, a cell to cell signaling mechanism mediated by small molecules that are produced by specific signal molecule synthases, to regulate gene expression in response to population density. In Vibrio anguillarum, the quorum-sensing phosphorelay channels information from three hybrid sensor kinases VanN, VanQ, CqsS that sense signal molecules produced by the synthases VanM, VanS and CqsA, onto the phosphotransferase VanU, to regulate activity of the response regulator VanO. VanO activates transcription of quorum-sensing regulatory RNAs (Qrr), which work together with the RNA chaperone Hfq to repress expression of the transcriptional regulator VanT.

The work presented in this thesis characterizes quorum-sensing independent and quorum-sensing dependent mechanisms that regulate VanT expression. Moreover, an in vivo imaging system was established, as a means to study V. anguillarum infections in the rainbow trout infection model.

Two quorum-sensing independent mechanisms regulating VanT expression were identified. First, the sigma factor RpoS indirectly activates VanT expression during transition into stationary growth phase by inhibiting hfq expression. Both, RpoS and VanT are crucial for stress response. Second, a type VI secretion system (T6SS) has a novel function as a signal sensing mechanism to regulate rpoS and vanT expression. Consequently, RpoS, quorum sensing and T6SS form a global network that senses stress and modulates stress response to ensure survival of the bacteria.

Further analysis of the quorum-sensing dependent regulation of VanT expression by the phosphorelay system revealed that four qrr genes are expressed continuously during growth. The phosphotransferase VanU is suggested to activate two response regulators, VanO and a predicted second response regulator. Activated VanO induces expression of the Qrr sRNAs, whereas, the predicted response regulator represses expression of the Qrr sRNAs. Thus, VanU has a pivotal role in the regulation of VanT expression. The signal synthase VanM and VanT form a regulatory loop, in which VanM represses VanT by inducing expression of the Qrr sRNAs and VanT directly activates vanM expression to repress its own expression. Moreover, Hfq destabilizes vanM mRNA, repressing vanM expression. VanT forms another regulatory loop with the transcriptional regulator LuxT, in which LuxT activates vanT expression and VanT directly represses luxT expression.

V. anguillarum is an opportunistic pathogen that causes vibriosis, a terminal hemorrhagic septicemia. The spatial and temporal progression of the infection was analyzed using the whole animal with an in vivo bioluminescent imaging method. Initial studies showed that colonization of the fish skin requires the siderophore, the RNA chaperone Hfq and the exopolysaccharide transport system, which protects against the innate immunity on the skin. Colonization of the fish skin is crucial for disease.

Place, publisher, year, edition, pages
Umeå: Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet) Umeå Universitet, 2010. 86 p.
Keyword
Vibrio anguillarum, quorum sensing, Type VI secretion, stress response, virulence, skin colonization
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-33269 (URN)978-91-7264-958-3 (ISBN)
Public defence
2010-05-18, NUS, byggnad 6L, Major Groove, Umeå Universitet, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-27 Created: 2010-04-20 Last updated: 2011-03-14Bibliographically approved

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Weber, BarbaraLindell, KristofferElQaidi, SamirMilton, Debra L

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