Facilitation of Direct Conditional Knockout of Essential Genes in Bacillus licheniformis DSM13 by Comparative Genetic Analysis and Manipulation of Genetic Competence

The genetic manageability of the biotechnologically importantBacillus licheniformis is hampered due to its poor transformability, whereasBacillus subtilis efficiently takes up DNA during genetic competence, a quorum-sensing-dependent process. Since the sensor histidine kinase ComP, encoded by a gene of the quorum-sensing modulecomQXPA ofB. licheniformis DSM13, was found to be inactive due to an insertion element withincomP , the coding region was exchanged with a functional copy. Quorum sensing was restored, but the already-poor genetic competence dropped further. The inducible expression of the key regulator for the transcription of competence genes, ComK, intrans resulted in highly competent strains and facilitated the direct disruption of genes, as well as the conditional knockout of an essential operon. As ComK is inhibited at low cell densities by a proteolytic complex in which MecA binds ComK and such inhibition is antagonized by the interaction of MecA with ComS (the expression of the latter is controlled by cell density inB. subtilis ), we performed anin silico analysis of MecA and the hitherto unidentified ComS, which revealed differences for competent and noncompetent strains, indicating that the reduced competence possibly is due to a nonfunctional coupling of thecomQXPA -encoded quorum module and ComK. The obtained increased genetic tractability of this industrial workhorse should improve a wide array of scientific investigations.