Cloning And Functional Analysis Of Quorum Sensing Gene ComQ In Bacillus Subtilis Strain NCD-2

The Bacillus subtilis NCD-2 is a good biocontrol agent against the cotton Verticillium wilt, its biocontrol effect could be enhanced by genetically modification. Transformation efficiency of B. subtilis strain NCD-2 was compared by using 5 different plasmids with various sizes and replication origins. The experimental results showed that no any correlation between transformation efficiency of strain NCD-2 and the plasmid size. For example, plasmid pNW33N carried the replication from B. coagulans showed the highest transformation efficiency of 3.6×10⁴ cfu·μg^-1 DNA. Other parameters affecting the transformation efficiency, including electric resistances, electric field strengths and cell growth phases, were also optimized with plasmid pNW33N. The result revealed that the highest transformation efficiency of 6.1×10⁴ cfu·μg^-1 DNA in strain NCD-2 could be obtained under the optimized condition, the cells of strain NCD-2 electroporated at 14.0 kV·cm^-1 and 200Ωafter incubated at 150 r·min^-1 and 37℃in growth medium for 3.5 h.Quorum-sensing (QS) system of bacteria is a mechanism that the bacteria produce and release chemical signal molecules whose external concentration increases as a function of increasing cell-population density. Bacteria detect the accumulation of a minimal threshold stimulatory concentration of these signals and alter gene expression. In B. subtilis, the formation of biofilm, the production of antibiotics and the sporulation are regulated under the QS system. Four genes including comX, comQ, comP and comA are included in the QS system in B. subtilis. ComX, a 10-amino acid peptide that is processed and secreted by ComQ, is detected by the membrane-bound histidine sensor kinase ComP, ComX binding stimulates ComP to autophosphorylate and transfer phosphate to the DNA-binding response regulator ComA. Phosphorylated ComA regulates transcription of a variety of genes .In this study, the QS system was identified from strain NCD-2 by scanning the whole genome sequences of strain NCD-2, and coloned the comX, comQ, comP and comA. The comQ gene was knockout by Homologous recombination in strain NCD-2, the mutant strain termed M6.The lipopeptides were extracted from the cultural supernatant of NCD-2 and M6. respectively, and the antifungal activities of lipopeptide extracted from M6 against the growth of Botrytis cinerea showed significant decreased compared to NCD-2 wild type strain. MALDI-TOF-MS analysis indicated that the production of FengycinB was decreased in the mutant strain M6, but the production of lipopeptide IturinB was increased when compared to the NCD-2 wild type strain. Indicated that the FengycinB are positively regulated by comQ and IturinB are negatively regulated by comQ. At the same time, some no reported substances were also detected both in NCD-2 wild type strain and the mutant strain M6, but the concentration was significantly decreased in the M6 strain compared to NCD-2 wild type strain.Compared the capability of biofilm formation between the NCD-2 wild type strain and M6 revealed that the biofilm in mutant M6 significant decreased, indicated that the biofilm formation are positively regulated by comQ.This study maybe provide an important basis for further understanding the function of quorum sensing system in B. subtilis NCD-2 strain biocontrol capability and its impact on the biocontrol activity, those results are useful for chaification the mechanism of B. subtilis strain NCD-2 suppression against the cotton Verticillium wilt.