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

RGP002Bistability in myo-inositol utilization by Salmonellaenterica serovar TyphimuriumT. Fuchs*Department of Microbiology, Central Institute for Food and NutritionResearch (ZIEL), Freising, GermanyThe capability of Salmonella enterica serovar Typhimurium to utilize myoinositol(MI) is determined by the genomic island GEI4417/4436 carryingthe iol genes. These encode enzymes, transporters and the repressor IolR.This autoregulated protein binds to four iol promoters and is released uponbinding of DKP, a metabolite of MI degradation . In contrast to all gramnegativeand gram-positive bacteria investigated so far, S. enterica serovarTyphimurium strain 14028 growing on MI as sole carbon source ischaracterized by a remarkable long lag phase of 40-60 hours. On solidmedium containing MI as sole carbon source, this human pathogen exhibitsa bistable phenotype characterized by a dissection into large colonies and aslow-growing bacterial background. This heterogeneity is reversible and notcaused by mutation. It is not observed in the absence of the iol generepressor IolR, nor in the presence of at least 0.55% CO 2. Upon analysis ofpromoter-gfp fusions, bistability could be linked to the activity of the iolEpromoter (P iolE) that is not controlled by IolR. On the single cell level,fluorescence microscopy and flow cytometry analysis revealed a gradualswitch of P iolE from the „off” to the „on” status during the late lag phase andthe transition to the log phase. Adding of ethoxyzolamide, an inhibitor ofcarboanhydrases, elongated the lag phase in the presence of bicarbonate. Thepositive feedback loop via repressor release and positive induction bybicarbonate/CO 2 might allow strain 14028 to adapt to rapidly changingenvironments. This is a novel example of bistability in substratedegradation, and, to our knowledge, the first example of gene regulation bybicarbonate/CO 2 in Salmonella.[1] Kröger, C. et al (2011): Hydrogen carbonate-dependent bistability in myo-inositol utilization bySalmonella enterica serovar Typhimurium. J. Bacteriol., in revision.[2] Kröger, C., and T. M. Fuchs (2010): Myo-Inositol transport by Salmonella enterica serovarTyphimurium. Microbiology 156, 128-138.[3] Kröger, C. and T. M. Fuchs (2009): Characterization of the myo-inositol utilization island ofSalmonella typhimurium. J. Bacteriol. 191, 545-554.RGP003Regulation of mitochondrial DNA inheritance andintegrity by the a2 mating-type locus genes lga2 and rga2of Ustilago maydisC. Basse* 1 , A. Pfeifer 1 , F. Nieto-Jacobo 2 , B. Martin 1 , D. Pasch 11 Institute for Applied Biosciences/ Genetics, Karlsruhe Institute ofTechnology (KIT), Karlsruhe, Germany2 Organismic Interactions, Max Planck Institute for TerrestrialMicrobiology, Marburg, GermanyThe Ustilago maydis a2 mating-type locus genes lga2 and rga2 play a rolein controlling uniparental mitochondrial DNA (mtDNA) inheritance duringthe sexual cycle. In particular, lga2 triggers selective loss of mtDNA of thea1 partner, while rga2 plays a role in protecting the a2-associated mtDNAfrom elimination. The mode of action of Lga2 and Rga2 is currently unclear,however, Lga2 likely acts by causing transient damage of unprotectedmitochondria. This is exemplified by large-scale transcriptional deregulationas well as efficient mitophagy in cells conditionally overexpressing lga2.Here, mitophagy, albeit controlled by atg8, follows a different inductionmechanism than under starvation conditions and involves the mitochondrialfission factor dnm1. Interference with mitochondrial fusion during mating isa major consequence of lga2 and efficiently constrains recombinationbetween parental mtDNAs. In this regard, we could provide evidence formitochondrial intron-encoded homing endonuclease activity and anunderlying role in promoting mtDNA recombination under conditions ofbiparental inheritance.RGP004Three distinct NssR-type regulators are involved intranscriptional control of Wolinella succinogenes geneclusters encoding reductases for nitrate, nitrite andnitrous oxideM. Kern*, J. SimonInstitute of Microbiology and Genetics, University of Technology,Darmstadt, GermanyEpsilonproteobacteria form a globally ubiquitous group of ecologicallysignificant organisms and comprise a diverse range of host-associated andfree-living bacteria. Many of these reduce nitrate to nitrite followed by eithernitrite ammonification or denitrification [1], but little is known aboutepsilonproteobacterial nitrosative stress defence, nitrogen compound sensingand the corresponding transcriptional regulation of respiratory enzymes.The model Epsilonproteobacterium Wolinella succinogenes uses the Nap,Nrf and cNos systems to reduce nitrate, nitrite or nitrous oxide (yieldingeither ammonium or dinitrogen) and all three enzyme systems areupregulated in the presence of nitrate or nitrous oxide. Typical NssR bindingsequences are present upstream of the transcriptional start sites of the nap,nrf and nos gene clusters and three distinct NssR-type regulators belongingto the Crp-Fnr-Dnr superfamily of transcription regulators are encoded onthe W. succinogenes genome. Corresponding gene deletion mutants wereconstructed and characterized with respect to anaerobic growth andinduction of the terminal reductases NapA, NrfA and cNosZ by variousnitrogen compounds.The experimental data indicate that all three NssR-type regulators arespecifically involved in respiratory nitrogen metabolism and/or nitrosativestress defence by activating nap, nrf and nos gene expression in response toeither nitrate, nitrous oxide or nitric oxide-induced stress.[1] Kern & Simon (2009): BBA 1787: 646-656.RGP005Characterization of the GlnR regulon in MycobacteriumsmegmatisN. Jeßberger*, A. BurkovskiInstitute for Microbiology, Friedrich-Alexander-Universiy, Erlangen,GermanyBased on sequence analyses and studies of a deletion mutant, the OmpRtyperegulator GlnR was recently identified as the transcriptional regulatorof nitrogen metabolism in Mycobacterium smegmatis [1, 2]. Transcriptionalregulation of the two target genes amtB (ammonium transporter) and glnA(glutamine synthetase) by GlnR was already shown, as well as binding ofthe regulator protein to the corresponding promoter regions [2].For further investigations, a global analysis method was chosen: geneexpression under nitrogen starvation was compared between the M.smegmatis wild type and a glnR deletion mutant in a DNA microarrayexperiment. 123 new putative GlnR target genes, including genes fordifferent ammonium transporters, glutamine synthetases, a nitrite reductionsystem, a methylamine oxidase, amidases, and purine and amino acidpermeases, were identified. These results were confirmed for more than 30genes in RNA hybridization experiments, where an expression of thesegenes depending on GlnR was observed. These data were validated for about20 genes in a second, independent approach, performing quantitative RTPCR. Binding of purified GlnR to promoter sequences of 13 target genes oroperons was also shown.Growth experiments with the M. smegmatis wild type strain and the glnRdeletion mutant were carried out using different new nitrogen sourcesindicated by the microarray data. Indeed, reduced or no growth of the glnRdeletion mutant was observed for about 10 of the tested nitrogen sources.All these data confirm the global role of GlnR as the main regulator ofnitrogen metabolism and its great influence on the expression of genesinvolved in uptake and assimilation of various nitrogen sources.[1] Amon, J.et al (2008): A genomic view on nitrogen metabolism and nitrogen control inmycobacteria. J. Mol. Microbiol. Biotechnol. 17(1):20-29.[2] Amon, J. et al (2008): Nitrogen control in Mycobacterium smegmatis: Nitrogen-dependentexpression of ammonium transport and assimilation proteins depends on OmpR-type regulator GlnR.J. Bacteriol. 190(21): 7108-7116.spektrum | Tagungsband 2011