Functional characterization of the Bacillus subtilis conjugative plasmid pLS20: identification of relaxosome components and mechanism of transcriptional control of main conjugation promoter Pc

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. The plasmid pLS20 is a native conjugative plasmid isolated from Bacillus subtilis “natto”. Our earlier laboratory results reported a global view of the regulatory system of the pLS20 conjugation genes. It was found that about 40 conjugation genes were regulated by a divergently oriented single gene rcoLS20, which encodes for the master regulator of conjugation. RcoLS20 is responsible for keeping the system in its default “OFF” state. Conjugation is activated by anti-­‐repressor RapLS20, which belongs of the family of Rap proteins. However, the activity of RapLS20 is regulated by a signaling peptide Phr*LS20. Even though, the key players involved in the regulation of the conjugation genes have been identified, our knowledge on regulation of the genetic switch responsible for activating conjugation is limited. In the first chapter of the thesis, we have studied in detail the molecular mechanism regulating the pLS20 conjugation genes using different in vivo and in vitro approaches. At least three levels of regulation controls plasmid mediated-­‐ conjugation including overlapping and divergent promoter of differing strengths, maintaining the expression of the genes of conjugation and repression, along with a tri-­‐functional repressor RcoLS20, which regulates its own transcription and finally as a tetramer RcoLS20, by DNA-­‐looping. These complex regulatory levels permits the switching “OFF” and “ON” of the system of conjugation. The second chapter of the thesis studies the formation of the relaxosome of plasmid pLS20. This nucleoprotein is essential during conjugation, permitting the transfer of DNA. In case of plasmid pLS20, we found that besides the relaxase, encoded by gene 58 of pLS20, and that we named RelLS20, the functional processing of oriT requires two additional proteins encoded by genes 56 and 57.