title: Induction of Plasmid Conjugation in Bacillus subtilis Is Bistable and Driven by a Direct Interaction of a Rap/Phr Quorum-sensing System with a Master Repressor.
abstract: Conjugation of plasmid pLS20 from Bacillus subtilis is limited to a time window between early and late exponential growth. Genetic evidence has suggested that pLS20-encoded protein RcoLS20 represses expression of a large conjugation operon, whereas Rap protein RapLS20 relieves repression. We show that RapLS20 is a true antirepressor protein that forms dimers in vivo and in vitro and that it directly binds to the repressor protein RcoLS20 in a 1:1 stoichiometry. We provide evidence that RapLS20 binds to the helix-turn-helix-containing domain of RcoLS20 in vivo, probably obstructing DNA binding of RcoLS20, as seen in competitive DNA binding experiments. The activity of RapLS20 in turn is counteracted by the addition of the cognate PhrLS20 peptide, which directly binds to the Rap protein and presumably induces a conformational change of the antirepressor. Thus, a Rap protein acts directly as an antirepressor protein during regulation of plasmid conjugation, turning on conjugation, and is counteracted by the PhrLS20 peptide, which, by analogy to known Rap/Phr systems, is secreted and taken back up into the cells, mediating cell density-driven regulation. Finally, we show that this switchlike process establishes a population heterogeneity, where up to 30% of the cells induce transcription of the conjugation operon.
authors.affiliation: From the LOEWE Zentrum für synthetische Mikrobiologie (SYNMIKRO), 35043 Marburg, Germany, the Spemann Graduate School of Biology and Medicine (SGBM), 79104 Freiburg, Germany, and the Fachbereich für Chemie, Hans-Meerwein Strasse, Universität Marburg, 35043 Marburg, Germany.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Rösch
authors.affiliation: From the LOEWE Zentrum für synthetische Mikrobiologie (SYNMIKRO), 35043 Marburg, Germany, the Fachbereich für Chemie, Hans-Meerwein Strasse, Universität Marburg, 35043 Marburg, Germany peter.graumann@synmikro.uni-marburg.de.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Graumann
mesh_terms: D001412:Bacillus subtilis / Q000502:physiology*; D003227:Conjugation, Genetic; D009876:Operon; D010957:Plasmids; D053038:Quorum Sensing; D012097:Repressor Proteins / Q000502:physiology*
chemical_list: D012097:Repressor Proteins
keywords: Bacillus subtilis; Rap-Phr module; bacterial conjugation; gene regulation; plasmid; protein-nucleic acid interaction; protein-protein interaction; quorum sensing