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

Abstract

The genome of Bacillus subtilis 168 encodes eight rap-phr quorum-sensing pairs. Rap proteins of all characterized Rap-Phr pairs inhibit the function of one or several important response regulators: ComA, Spo0F, or DegU. This inhibition is relieved upon binding of the peptide encoded by the cognate phr gene. Bacillus subtilis strain NCIB3610, the biofilm-proficient ancestor of strain 168, encodes, in addition, the rapP-phrP pair on the plasmid pBS32. RapP was shown to dephosphorylate Spo0F and to regulate biofilm formation, but unlike other Rap-Phr pairs, RapP does not interact with PhrP. In this work we extend the analysis of the RapP pathway by reexamining its transcriptional regulation, its effect on downstream targets, and its interaction with PhrP. At the transcriptional level, we show that rapP and phrP regulation is similar to that of other rap-phr pairs. We further find that RapP has an Spo0F-independent negative effect on biofilm-related genes, which is mediated by the response regulator ComA. Finally, we find that the insensitivity of RapP to PhrP is due to a substitution of a highly conserved residue in the peptide binding domain of the rapP allele of strain NCIB3610. Reversing this substitution to the consensus amino acid restores the PhrP dependence of RapP activity and eliminates the effects of the rapP-phrP locus on ComA activity and biofilm formation. Taken together, our results suggest that RapP strongly represses biofilm formation through multiple targets and that PhrP does not counteract RapP due to a rare mutation in rapP.T he behavior of bacterial communities is often regulated by quorum-sensing (QS) signaling pathways. In these pathways, a secreted molecular signal accumulates in the environment and activates a cognate receptor at sufficiently high concentrations. Gram-positive bacteria frequently utilize peptides as QS signals (1-3), thereby eliciting a variety of behaviors, including the secretion of various molecules (e.g., enzymes, antibiotics, surfactants, and exopolysaccharides [4]), the initiation of developmental processes (such as sporulation and biofilm formation), and horizontal gene transfer through transformation or conjugation (5-7).Members of the Rap-Phr family of QS systems in the Grampositive model Bacillus subtilis are involved in the regulation of competence, sporulation, and biofilm formation. The chromosome of strain B. subtilis 168 encodes eight full receptor signal systems and three orphan receptors from this family (6,8). In each of the full systems, the phr gene encodes a prepeptide, which is secreted through the major secretory system and then further processed outside the cell, resulting in the formation of a mature penta-or hexapeptide signal (9). The mature peptide is transported into the cell by the oligopeptide-permease (Opp) complex, where it interacts with its Rap targets within the cytoplasm (10). All characterized Rap proteins contain two domains (10-12), a tricopeptide repeat (TPR) domain that interacts with the signaling peptide and a second domain that f...

Facultative cheating supports the coexistence of diverse quorum-sensing alleles

“…S4). The reduced relative fitness most likely reflects the lower levels of cooperative investment observed in this genetic background (32). Together, our results suggest that the quorum-sensing response, and specifically the production of surfactin during swarming, is a costly cooperative behavior and .…”

“…The domestication of the laboratory strain has been accompanied by a multitude of mutations and the loss of additional traits and regulatory circuits in addition to those related to swarming, such as the ability to form biofilms (31). To verify that these additional mutations do not significantly affect the social interactions between ComQXPA variants, we repeated some of the experiments in a biofilm-forming isolate (32). We find that in this genetic background, the ΔcomA and ΔcomQXP mutants exploited the wild type, as was observed in the laboratory strain background, albeit with a lower relative fitness (Fig.…”

Transient Duplication-Dependent Divergence and Horizontal Transfer Underlie the Evolutionary Dynamics of Bacterial Cell–Cell Signaling

“…The different Rap proteins were assayed for their effects on the Spo0A and ComA pathways by measuring their impact on sporulation efficiency and expression of the ComA-regulated srfA promoter using a yellow fluorescent protein (YFP) reporter [43], respectively (Fig 3A, S5 Data, Methods). Because ComA activity is indirectly affected by the Spo0A pathway [56], we introduced a spo0A deletion into each of the YFP-reporting strains.…”

A DegU-P and DegQ-Dependent Regulatory Pathway for the K-state in Bacillus subtilis

“…Another strain difference is that 3610 carries a large plasmid, which encodes RapP, a phosphatase that acts on Spo0F-P (Parashar et al, 2013). It has been reported that the inactivation of rapP mitigates the sharp downturn in srfA transcription (Parashar et al, 2013; Omer Bendori et al, 2015). This result has been verified in our hands; we have consistently observed a shoulder in the srfA transcription rate curves in the rapP knockout strain, and we have found that the transcription rate does not drop to zero as it does in the rapP + parent (Supplementary Figure S6A).…”