A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

B. subtilis biofilm formation is tightly regulated by elaborate signaling pathways. In contrast to domesticated lab strains of B. subtilis, which form smooth, essentially featureless colonies, undomesticated strains such as NCIB3610 form architecturally complex biofilms.  NCIB3610 also encodes an 80-kb plasmid absent from laboratory strains, and mutations in a plasmid-encoded homolog of a Rap protein, RapP, caused a hyper-rugose biofilm phenotype.

Here we explored the role of rapP-phrP in biofilm formation. We found that RapP is a phosphatase that dephosphorylates the intermediate response regulator Spo0F. RapP appears to employ a catalytic glutamate to dephosphorylate the Spo0F aspartylphosphate, and the implications of the RapP catalytic glutamate are discussed. In addition to regulating B. subtilis biofilm formation, we found that RapP regulates sporulation and genetic competence as a result of its ability to dephosphorylate Spo0F. Interestingly, while rap-phr gene cassettes routinely form regulatory pairs, i.e., the mature phr gene product inhibits the activity of the rap gene product, the phrP gene product did not inhibit RapP activity in our assays. RapP activity was, however, inhibited by PhrH in vivo but not in vitro.

Additional genetic analysis suggests that RapP is directly inhibited by peptide binding. We speculate that PhrH could be subject to post-translational modification in vivo and directly inhibit RapP activity, or, more likely, PhrH upregulates the expression of a peptide that in turn directly binds to RapP and inhibits its Spo0F phosphatase activity.

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