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.

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

“…Previous work has shown that Rco regulates transcription of the conjugation operon through binding to two operator sites within the intergenic region of rco and the first gene of the conjugation operon, possibly by forming a DNA loop of 75 bp (19). Although we did not observe a preference of the repressor to form tetramers as stated before, we found a DNA binding behavior comparable with that described in the literature (Fig.…”

“…3, first panel). However, binding affinity was lower in this region compared with binding to the previously described operator binding sites (19) (Fig. 2D), suggesting that the newly identified binding region may act as a secondary repressor binding site.…”

“…Interestingly, time lapse microscopy revealed that once the ON state is established, expression of the conjugation promoter is propagated to the next generation of cells and vanishes with additional cell divisions. Interestingly, the Meijer group (19) recently showed that repressor protein Rco LS20 autoregulates itself and keeps the conjugation operon in the OFF state through binding to two operator sites, presumably forming a DNA loop. Loop formation may contribute to the relatively tight on/off regulation seen in our expression studies.…”

“…We have found that this is also the case for strain PY79 (data not shown), confirming that the protein acts as a repressor independent of strain background. Ramachandran et al (19) identified two convergent and overlapping promotors that drive the expression of the repressor gene and the conjugation operon. In silico analysis of the intergenic region between the first gene of the conjugation operon and the repressor gene using the DBTBS database (20) revealed a sigW promoter-like sequence 27 bp upstream of the rco gene (5Ј-cGAAAa-N 16 -CGTATA).…”

Mechanisms of peptide sex pheromone regulation of conjugation in Enterococcus faecalis

“…Recently, Ramachandran et al. (Ramachandran et al., ) reported the existence of a DNA loop in the conjugative pLS20 plasmid of the soil bacterium Bacillus subtilis . Similar to pCF10, transcriptional regulation of pLS20 major conjugation operon is repressed by the master repressor protein Rco LS20 .…”