VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

RGP043Influence of Temperature on expression and stability ofthe RovA/SlyA regulator familyC. Mendonca* 1 , K. Herbst 1 , N. Quade 2 , A.K. Heroven 1 , P. Dersch 11 Helmholtz Center for Infection Research, Molecular Infection Biology,Braunschweig, Germany2 Helmholtz Center for Infection Research, Braunschweig, GermanyTranscriptional regulation of genes under a specific set of conditions is away in which bacteria adapt to a variety of environmental conditions. Theregulatory proteins SlyA of Salmonella typhimurium and RovA of Yersiniapseudotuberculosis belong to the MarR regulator family and control severalphysiological processes relevant for virulence and survival. The RovA/SlyAproteins are very closely related in sequence. The helix-turn-helix DNAbinding sites of Salmonella SlyA and the Yersinia RovA protein are almostidentical but they control largely different gene sets, reflecting bothregulation of species-specific targets and transcriptional rewiring of sharedgenes. SlyA of Salmonella was shown to interact with the stringent controlsignal molecule ppGpp, which enhanced its DNA-binding activity. Incontrast, RovA acts as an intrinsic thermometer that undergoes structuralalterations in response to a temperature shift from 25°C to 37°C. At 37°CRovA is rapidly degraded by the Lon protease. In contrast, proteindegradation assays carried out at 25°C and 37°C demonstrated that SlyA ofSalmonella is stable at both temperatures. To further investigate thethermosensor in RovA, amino acid exchanges using SlyA as a template wereintroduced into a Plac-driven RovA expression system. Using this strategywe were able to identify certain amino acids which render RovA resistant totemperature-induced degradation at 37°C. Furthermore, we found that in Y.pseudotubercuolsis, in stationary phase a factor is secreted into the growthmedium. This factor completely stabilises RovA at 37°C. Currentexperiments are directed to identify this RovA-stabilising factor.RGP044The novel PAS4-LuxR solo Plu2018/Plu2019 of the insectpathogen Photorhabdus luminescens detects a eukaryoticsignaling moleculeE. Rothmeier, C. Manske, R. Heermann*Department of Microbiology, Ludwig-Maximillians-Universtiy, Munich,GermanyIn nature, bacteria live in close association with other bacteria andeukaryotes which means that they constantly need to monitor andcommunicate with other organisms. The best understood chemical languagein proteobacteria is the communication via N-acylhomoserine lactones(AHLs), often produced as an endogenous signal and called quorum sensing.The typical proteobacterial quorum sensing system consists of an AHLsynthase belonging to the LuxI-family and a cognate LuxR-family AHLsensor/regulator. Many proteobacteria possess further LuxR-family proteinswith no cognate LuxI synthase. Initial investigations of those so called LuxRsolos revealed that these regulators have diverse roles in bacteriainterspecies and interkingdom communication. The insect pathogenicbacterium Photorhabdus luminescens possesses the uncommonly highnumber of 39 LuxR solos, 35 of them have a novel PAS4 signal bindingdomain. These PAS4-LuxR solos are speculated to detect yet unknowneukaryotic signaling molecules. Most of the corresponding genes of thePAS4-LuxR solos are located within two large gene clusters on the P.luminsescens chromosome. Here, we inactivated these large PAS4-luxR geneclusters and performed proteome analyses with the mutants in comparison tothe wild-type with filtered insect homogenate as putative inducer. Thisallowed the identification of potential target genes of these regulators and,on the basis of this knowledge, the generation of corresponding reportergene strains. We could show that the expression of several reporter geneswas inducible with insect homogenate in the wild-type, but not in the mutantlacking PAS4-LuxR solos Plu2018/Plu2019. This clearly showed that thesenovel PAS4-LuxR solos are involved in interkingdom signaling in P.luminescens. Stability experiments with the insect homogenate revealed thatthe signaling molecule sensed by Plu2018/Plu2019 could be a hormone-likesubstance.RGP045Characterisation of furA expression in Mycobacteriumavium spp. paratuberculosisT. Meißner*, J. Meens, G.-F. Gerlach, R. GoetheInstitute for Microbiology, University of Veterinary Medicine, Hannover,GermanyMycobacterium avium spp. paratuberculosis (MAP) is the etiological agentof paratuberculosis (Johne´s disease), a chronic, incurable, granulomatousenteritis in ruminants. Furthermore a contribution of MAP to human Crohn´sdisease is discussed.In the host MAP has to overcome the iron starvation by expressing ferricuptake systems via iron depending regulators. The ferric uptake regulator A(FurA), a homolog to the ferric uptake regulator (Fur) family, is animportant regulator of iron homeostasis in many bacteria includingmycobacteria. Only little is known about iron dependent regulation in MAP.The iron dependent regulator (IdeR) belonging to the diphtheria toxinregulator (DtxR) family has shown to be responsible for iron mediated generegulation. IdeR is essential for the expression of a cohort of genes encodingproteins for iron uptake and storage. However, the function and regulation ofFurA in MAP is still unknown.By analysing the MAP DSMZ44135 genome the position of the furA genewas detected close to the katG gene encoding a catalase-peroxidase KatG.Both genes were expressed on a polycistronic RNA. The furA-katG region ishighly conserved among the order actinomycetales and it was shown to beinduced and expressed under oxidative stress and iron starvation.Additionally it has been demonstrated, that FurA auto-regulates its ownexpression in Mycobacterium tuberculosis. In the present study, we culturedMAP in the presence of dipyridyl, an iron chelating agent. Theseexperiments revealed that in MAP furA mRNA expression is not inducibleby iron starvation, while IdeR dependent genes were up-regulated. Thesedata suggest that MAP furA is not auto-regulatory or dependent on otherdivalent cations.Genetic manipulation of MAP is hampered by its slow growth and clumpformation. Therefore, in order to analyse the role of FurA more in detail, weapplied the specialized transduction method for furA deletion. Fortransduction, we use the pHAE87 phage, a temperature-sensitive derivate ofthe TM4 mycobacteriophage and constructed a new Phage (pHAE151) toexchange the furA gene with a hygromycin resistance gene.RGP046An RpoS-dependent small RNA controls OmpD proteinsynthesis in SalmonellaK. Fröhlich*, K. Papenfort, J. VogelRNA Biology, Institute for Molecular Infection Biology (IMIB), Würzburg,GermanyQuestion: Small non-coding RNAs (sRNA) are a steadily growing class ofpost-transcriptional regulators frequently involved in bacterial stressresponses. While the transcription of two stationary phase-specific sRNAs,RybB and MicA, was reported to be tightly controlled by the alternativesigma factor, σ E, no sRNA has yet been assigned to the regulon of the majorstress sigma factor σ S (RpoS).Methods: In a genome-wide transposon screen we discovered thealternative sigma factor S as the direct transcriptional regulator of theconserved sRNA, SdsR. Over-expression of the sRNA readily inhibited theexpression of the abundant outer membrane protein OmpD.Results: We identified a highly conserved sRNA, SdsR, which accumulatesin high amounts in stationary phase and is transcriptionally dependent onRpoS. In Salmonella, SdsR represses the expression of the porin OmpDthrough direct base-pairing. Similar to an additional regulatory RNA, MicC,SdsR binds within the coding sequence of ompD mRNA and down-regulationrequires both the presence of the RNA chaperone Hfq as well as RNaseE.Conclusions: In this study we report the characterization of a non-codingRNA, SdsR, as the first sRNA directly controlled by the alternative sigmafactor σ S. Over-expression of SdsR in Salmonella reduced the expression ofthe OmpD protein. SdsR-mediated repression of ompD requires binding inthe coding sequence suggesting a mechanism independent of inhibition oftranslation initiation.spektrum | Tagungsband 2011