ComX-Induced Exoproteases Degrade ComX in Bacillus subtilis PS-216

Mihael Spacapan, Tjaša Danevčič, Ines Mandic-Mulec

Gram-positive bacteria use peptides as auto-inducing (AI) signals to regulate the production of extracellular enzymes (e.g., proteases). ComX is an AI peptide, mostly known for its role in the regulation of bacterial competence and surfactant production inBacillus subtilis. These two traits are regulated accordingly to the bacterial population size, thus classifying ComX as a quorum sensing signal. ComX also indirectly regulates exoprotease production through the intermediate transcriptional regulator DegQ. We here use this peptide-based AI system (the ComQXPA system) as a model to address exoprotease regulation by ComX in biofilms. We also investigate the potential of ComX regulated proteases to degrade the ComX AI peptide. Results indicate that ComX indeed induces the expression ofaprE, the gene for the major serine protease subtilisin, and stimulates overall exoprotease production in biofilms ofB. subtilisPS-216 and several otherB. subtilissoil isolates. We also provide evidence that these exoproteases can degrade ComX. The ComX biological activity decay is reduced in the spent media of floating biofilms with low proteolytic activity found in thecomPanddegQmutants. ComX biological activity decay can be restored by the addition of subtilisin to such media. In contrast, inhibition of metalloproteases by EDTA reduces ComX biological activity decay. This suggests that both serine and metalloproteases, which are induced by ComX, are ultimately capable of degrading this signaling peptide. This work brings novel information on regulation of exoproteases inB. subtilisfloating biofilms and reveals that these proteolytic enzymes degrade the AI signaling peptide ComX, which is also a major determinant of their expression in biofilms.