What renders Bacilli genetically competent? A gaze beyond the model organism.

Natural genetic competence enables bacteria to take in and establish exogenously supplied DNA and thus constitutes a valuable tool for strain improvement. Extensively studied in the Gram-positive model organism Bacillus subtilis genetic competence has indeed proven successful for genetic manipulation aiming at enhancement of handling, yield, and biosafety. The majority of Bacilli, particularly those relevant for industrial application, do not or only poorly develop genetic competence, although rather homologous DNA-uptake machineries are routinely encoded. Establishing the competent state solely due to high cell densities (quorum sensing dependency) appears to be restricted to the model organism, in which the small signalling peptide ComS initiates the regulatory pathway that ultimately leads to the expression of all genes necessary for reaching the competent state. Agreeing with the lack of a functional ComS peptide, competence-mediated transformation of other Bacilli depends on nutrient exhaustion rather than cell density. Genetically, competent strains of the model organism B. subtilis, cultivated for a long time and selected for laboratory purposes, display probably not least to such selection a point mutation in the promoter of a regulatory gene that favors competence development whereas the wild-type progenitor only poorly displays genetic competence. Consistent with competence being a matter of deregulation, all strains of Bacillus licheniformis displaying efficient DNA uptake were found to carry mutations in regulator genes, which are responsible for their genetic competence. Thus, strain-specific genetic equipment and regulation as well as the proven role of domestication for the well-established laboratory strains ought to be considered when attempting to broaden the applicability of competence as a genetic tool for strains other than the model organism.

The two putative comS homologs of the biotechnologically important Bacillus licheniformis do not contribute to competence development.

In Bacillus subtilis, natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating-among others-DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis, ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis. Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis. Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis.

Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains.

Bacterial natural genetic competence - well studied in Bacillus subtilis - enables cells to take up and integrate extracellularly supplied DNA into their own genome. However, little is known about competence development and its regulation in other members of the genus, although DNA uptake machineries are routinely encoded. Auxotrophic Bacillus licheniformis 9945A derivatives, obtained from repeated rounds of random mutagenesis, were long known to develop natural competence. Inspection of the colony morphology and extracellular enzyme secretion of two of these derivatives, M28 and M18, suggested that regulator genes are collaterally hit. M28 emerged as a 14 bp deletion mutant concomitantly displaying a shift in the reading frame of degS that encodes the sensor histidine kinase, which is part of the molecular switch that directs cells to genetic competence, the synthesis of extracellular enzymes or biofilm formation, while for M18, sequencing of the suspected gene revealed a 375 bp deletion in abrB, encoding the major transition state regulator. With respect to colony morphology, enzyme secretion and competence development, both of the mutations, when newly generated on the wild-type B. licheniformis 9945A genetic background, resulted in phenotypes resembling M28 and M18, respectively. All of the known naturally competent B. licheniformis representatives, hitherto thoroughly investigated in this regard, carry mutations in regulator genes, and hence genetic competence observed in domesticated strains supposedly results from deregulation.