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 important Bacillus licheniformis is hampered due to its poor transformability, whereas Bacillus 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 module comQXPA of B. licheniformis DSM13, was found to be inactive due to an insertion element within comP, 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, in trans 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 in B. subtilis), we performed an in 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 the comQXPA-encoded quorum module and ComK. The obtained increased genetic tractability of this industrial workhorse should improve a wide array of scientific investigations.
Hoffmann
Kerstin
K
Westfälische Wilhelms-Universität Münster, Institut für Molekulare Mikrobiologie und Biotechnologie, Corrensstrasse 3, 48149 Münster, Germany.
Wollherr
Antje
A
Larsen
Michael
M
Rachinger
Michael
M
Liesegang
Heiko
H
Ehrenreich
Armin
A
Meinhardt
Friedhelm
F
D001407:Bacillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D020043:Genes, Essential; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D053038:Quorum Sensing; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014157:Transcription Factors; C087449:comK protein, Bacillus subtilis