title: A complex genetic switch involving overlapping divergent promoters and DNA looping regulates expression of conjugation genes of a gram-positive plasmid.
abstract: Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Gayetri
authors.initials: G
authors.lastname: Ramachandran
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Singh
authors.affiliation: Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
authors.forename: Juan Roman
authors.initials: JR
authors.lastname: Luque-Ortega
authors.affiliation: Centro Nacional de Biotecnología (CSIC), Canto Blanco, Madrid, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Yuste
authors.affiliation: Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Alfonso
authors.affiliation: Centro Nacional de Biotecnología (CSIC), Canto Blanco, Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Rojo
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle Upon Tyne, United Kingdom.
authors.forename: Ling J
authors.initials: LJ
authors.lastname: Wu
authors.affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Universidad Autónoma, Canto Blanco, Madrid, Spain.
authors.forename: Wilfried J J
authors.initials: WJ
authors.lastname: Meijer
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D003227:Conjugation, Genetic; D004352:Drug Resistance, Microbial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic