Novel mechanisms of controlling the activities of the transcription factors Spo0A and ComA by the plasmid‐encoded quorum sensing regulators Rap60‐Phr60 in Bacillus subtilis

Abstract

Summary Bacillus subtilis and its closest relatives have multiple rap-phr quorum sensing gene pairs that coordinate a variety of physiological processes with population density. Extra-chromosomal rap-phr genes are also present on mobile genetic elements, yet relatively little is known about their function. In this work, we demonstrate that Rap60-Phr60 from plasmid pTA1060 coordinates a variety of biological processes with population density including sporulation, cannibalism, biofilm formation and genetic competence. Similar to other Rap proteins that control sporulation, Rap60 modulates phosphorylation of the transcription factor Spo0A by acting as a phosphatase of Spo0F~P, an intermediate of the sporulation phosphorelay system. Additionally, Rap60 plays a noncanonical role in regulating the autophosphorylation of the sporulation-specific kinase KinA, a novel activity for Rap proteins. In contrast, Rap proteins that modulate genetic competence interfere with DNA binding by the transcription factor ComA. Rap60 regulates the activity of ComA in a unique manner by forming a Rap60–ComA–DNA ternary complex that inhibits transcription of target genes. Taken together, this work provides new insight into two novel mechanisms of regulating Spo0A and ComA by Rap60 and expands our general understanding of how plasmid-encoded quorum sensing pairs regulate important biological processes.

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

“…We show that the repressor and its antirepressor preferentially form dimers in solution and that complex formation occurs in a 1:1 stoichiometry that disrupts DNA binding of the repressor protein through obscuring the DNA-binding domain of the repressor. Unlike the plasmid-encoded Rap60, which presumably inactivates ComA activity by occluding its interaction with the RNA polymerase (26), inactivation of the Rco repressor by Rap LS20 resembles that of RapC, -F, and -H, which prevent DNA binding of ComA, which, however, is an activator, rather than a repressor, like Rco. Structural analysis of the RapF-ComA interaction showed that the N-terminal 3-helix bundle of RapF captures the helix-turnhelix-containing domain of ComA and thereby prevents binding of ComA to its target promoters (8).…”

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

“…RapF blocks comA binding to DNA through direct competition for the DNA binding domain [27]. In contrast, Rap60 does not block ComA DNA binding but prevents the DNA-bound ComA from activating transcription [46]. …”

Characterization of a Novel Iron Acquisition Activity That Coordinates the Iron Response with Population Density under Iron-Replete Conditions in Bacillus subtilis

“…Clones were verified by DNA sequencing (UMASS Core Facility). Plasmid DNA was linearized by digestion with NcoI restriction enzyme (NEB) and transformed into B. subtilis strain JH642 by a two-step procedure described by Boguslawski et al (50), and the strain was plated on LB solid medium with neomycin.…”

“…Liquid cultures were grown in S7 defined minimal medium salts (45) containing 50 mM 4-morpholinepropanesulfonic acid instead of 100 mM (S7 50 ) and supplemented with 1% glucose, 0.1% glutamate, and the following trace metals: 2 M MgCl 2 , 0.7 M CaCl 2 , 50 M MnCl 2 , 1 M ZnCl 2 , 1 g/ml of thiamine, and 5 M FeCl 3 . Solid medium plates contained Spizizen salts (46) supplemented with 1% glucose and 0.1% glutamate.…”