@article{RademacherHoffmannLackmannetal.2012, author = {Rademacher, Corinna and Hoffmann, Marie-Christine and Lackmann, Jan-Wilm and Moser, Roman and Pf{\"a}nder, Yvonne and Leimk{\"u}hler, Silke and Narberhaus, Franz and Masepohl, Bernd}, title = {Tellurite resistance gene trgB confers copper tolerance to Rhodobacter capsulatus}, series = {BioMetals : an international journal on the role of metal ions in biology, biochemistry and medicine}, volume = {25}, journal = {BioMetals : an international journal on the role of metal ions in biology, biochemistry and medicine}, number = {5}, publisher = {Springer}, address = {Dordrecht}, issn = {0966-0844}, doi = {10.1007/s10534-012-9566-2}, pages = {995 -- 1008}, year = {2012}, abstract = {To identify copper homeostasis genes in Rhodobacter capsulatus, we performed random transposon Tn5 mutagenesis. Screening of more than 10,000 Tn5 mutants identified tellurite resistance gene trgB as a so far unrecognized major copper tolerance determinant. The trgB gene is flanked by tellurite resistance gene trgA and cysteine synthase gene cysK2. While growth of trgA mutants was only moderately restricted by tellurite, trgB and cysK2 mutants were severely affected by tellurite, which implies that viability under tellurite stress requires increased cysteine levels. Mutational analyses revealed that trgB was the only gene in this chromosomal region conferring cross-tolerance towards copper. Expression of the monocistronic trgB gene required promoter elements overlapping the trgA coding region as shown by nested deletions. Neither copper nor tellurite affected trgB transcription as demonstrated by reverse transcriptase PCR and trgB-lacZ fusions. Addition of tellurite or copper gave rise to increased cellular tellurium and copper concentrations, respectively, as determined by inductively coupled plasma-optical emission spectroscopy. By contrast, cellular iron concentrations remained fairly constant irrespective of tellurite or copper addition. This is the first study demonstrating a direct link between copper and tellurite response in bacteria.}, language = {en} } @article{GisinMuellerPfaenderetal.2010, author = {Gisin, Jonathan and Mueller, Alexandra and Pfaender, Yvonne and Leimk{\"u}hler, Silke and Narberhaus, Franz and Masepohl, Bernd}, title = {A Rhodobacter capsulatus member of a universal permease family imports molybdate and other oxyanions}, issn = {0021-9193}, doi = {10.1128/Jb.00742-10}, year = {2010}, abstract = {Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.}, language = {en} } @article{WiethausMuellerNeumannetal.2009, author = {Wiethaus, Jessica and Mueller, Alexandra and Neumann, Meina and Neumann, Sandra and Leimk{\"u}hler, Silke and Narberhaus, Franz and Masepohl, Bernd}, title = {Specific interactions between four Molybdenum-binding proteins contribute to Mo-dependent gene regulation in Rhodobacter capsulatus}, issn = {0021-9193}, doi = {10.1128/Jb.00526-09}, year = {2009}, abstract = {The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA- binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.}, language = {en} }