Mobility of the Native Bacillus subtilis Conjugative Plasmid pLS20 Is Regulated by Intercellular Signaling

Abstract

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default “OFF” state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

The RapP-PhrP Quorum-Sensing System of Bacillus subtilis Strain NCIB3610 Affects Biofilm Formation through Multiple Targets, Due to an Atypical Signal-Insensitive Allele of RapP

“…Since srfA expression is modulated by multiple QS systems whose functions are not well characterized in strain 3610, we examined whether this repression is conserved when RapP is expressed in the genetic background of the lab strain PY79 in which QS is well characterized. The use of strain PY79 also allowed us to ignore interactions of RapP with plasmid-borne genes (as described with other Rap proteins [21,37]). In addition, the activity could be assessed without the indirect effects that surfactin may have on gene expression (38).…”

“…Finally, RapG represses phosphorylated DegU (DegUϳP) (19), whose main functions are to promote the secretion of multiple enzymes and repress social motility (20). Recently, it was hypothesized that a plasmid-borne Rap (specifically, Rap carried by plasmid pLS20 [Rap pLS20 ]) may also interact directly with an Xre-type plasmidborne repressor (21).…”

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

“…Rap LS20 Binds Directly to Rco to Inhibit Its DNA Binding Activity, whereas the Addition of Phr LS20 Relieves the Inhibitory Function of Rap LS20 on Rco-Genetic evidence has shown that an endogenous Rap-Phr module on pLS20 (Rap LS20 and Phr LS20 ) affects the activity of the repressor of conjugation (10). To determine whether Rap LS20 directly regulates the activity of the repressor protein through protein-protein interactions, we performed analytical size exclusion chromatography of Rap LS20 , Rco, and a mixture of both proteins.…”

“…Recently, it was found that a Rap/Phr-like module also plays a role in the regulation of transfer of the conjugative plasmid pLS20 from B. subtilis natto (10), which shows an unusual activity for conjugation; whereas ICEBs1 becomes active during the transition from exponential growth to stationary phase (11), pLS20 conjugates with some delay after cells have been resuspended into fresh medium and shuts down DNA transfer during late exponential phase, well before cells cease to grow (12). Interestingly, several components of the conjugation machinery of pLS20 assemble and disassemble at a single cell pole or a single site at the lateral membrane, in parallel with conjugation activity (13).…”

“…Interestingly, several components of the conjugation machinery of pLS20 assemble and disassemble at a single cell pole or a single site at the lateral membrane, in parallel with conjugation activity (13). How these kinetics are achieved has been hinted at through the identification of a repressor-like protein, Rco LS20 , whose deletion results in higher and constitutive conjugation activity, whereas its overproduction leads to a reduction in conjugation efficiency (10). Induction of pLS20 conjugation is also achieved through overexpression of a plasmid-encoded Rap protein, Rap LS20 , which may be counteracted by the expression of its cognate peptide Phr LS20 , encoded just downstream of the Rap protein-encoding gene.…”