Characterization of a native plasmid from Bacillus subtilis with special focus on its regulatory circuit for conjugation

Abstract

Spreading of antibiotic resistance among pathogenic bacteria is a major problem for human Health as well as in veterinarian world. Bacteria can exchange antibiotic resistance genes by Different processes, collectively called Horizontal Gene Transfer (HGT). One of the processes Of HGT is conjugationin which genetic material (DNA) is transferred from a donor cell to a Recipient cell. Conjugation, which is widespread among both Gram-negative and Gram- positive Bacteria is mediated by conjugation system present on plasmids and/or! by integrative conjugative elements (ICEs). Plasmids are autonomously replicating units present in many bacteria besides their chromosome. Conjugation systems present on various plasmids from Gram-negative bacteria have been studied in considerable detail. Much less is known, however, about conjugation !systems present!on plasmids from Gram- Positive bacteria. A major goal of my thesis studies was to increase ou understandingabout Conjugation systems and Its regulation in Gram-positive bacteria. We chose the! Native plasmid pLS20 from! Bacillus subtilis for several reasons. First; B. subtilis, itself a GRAS organism, is related to pathogens or fastidious bacteria, 2nd, is one of bes ! studied organisms, 3rd, its natural competence facilitates genetic manipulation, and 4th, B.subtilis is gut commensal. The first part of this thesis describes an in-silico analysis of pLS20cat, which we have Sequenced to completeness.pLS20cat has a size of 65,774 bps. It contains almost 100 ORFs, Which we have grouped into five different modules. One of the modules concerns a putative conjugation operon, which flanks a rap-phr cassette. Many rap-phr cassettes have been identified on the genome of B.( subtilis and other bacilli. In addition, they are present on several rolling-circle and theta replicating plasmids of Bacilli. Most of the rap-phr cassettes analyzed have been shown to affect differentiation processes like sporulation, competence and biofilm formation. A detailed analysis of the rap-phr cassette of pLS20cat is described in the third part of this thesis. We found that the presence of pLS20cat strongly inhibits transformation efficiencies Of its host. The second part of this thesis describes the identification and functional analysis of the pLS20cat-located gene responsible for competence inhibition. Thus, we found that ORF64 is responsible for competence inhibition and we named the gene rokLS20. We also unraveled the mechanism of how RokLS20 inhibits competence. RokLS20 is a DNA binding protein that preferentially binds to the comK promoter thereby repressing expression of the key transcriptional activator of the competence genes, ComK. A functional RokLS20IGFP fusion protein coIlocalized with the bacterial nucleoid in an irregular pattern similar to that described for the RokBs168 homologue that is encoded by the host. Screening the sequences present in available databases resulted in the identification of several additional! rok paralogues. Based on their homology, 20 rok genes were divided into twogroups. The third part of the thesis describes the regulatory circuit of pLS20cat conjugation. Our results show that the conjugation genes of pLS20cat are not induced by recipient produced pheromones, demonstrating that the conjugation genes of pLS20cat are fundamentally different regulated to those present on the Gram-positive enterococcal plasmids pAD1 and pCF10. Our results show that conjugation is in a!default! “OFF” state. Conjugation is activated by RapLS20, which is an anti-repressor and hence relieves RcoLS20-mediated repression. Finally,the phrLS20 encodes for the signaling peptide Phr*LS20 that !is responsible for inhibiting the activity of RapLS20 and decide the timing for conjugation.