Cloning Of The Lecithin-Solubilizing Related Gene And Identification Of Antifungal Substances From Bacillus Subtilis NCD-2

Bacillus subtilis strain NCD-2 was isolated from the rhizosphere of cotton, and showed antagonism to Verticillium dahliae by secreting antifungal active compounds. This strain also showed lecithin-solubilizing ability.Plasmid pHV1249 which carrying transposon mini-Tn10 was transferred into strain NCD-2 by electrotransformation, and the transposon library of NCD-2 was constructed. Three mutants with defective of lecithin-solubilizing ability were selected from more than 3000 mini-Tn10 inserted mutants. Southern hybridization with mini-Tn10 fragment as a probe showed that all these mutants contained single copy transposon insertion, which possibly localized at the same chromosomal location. One mutant M216 was used for further study. The flanking sequence of insertion site was cloned from mutant M216 by Nest PCR. Sequence analysis showed that the inserted gene has of 98% identity with phoR in B. subtilis strain 168. Genetic complementation was conducted by cloning phoR gene into plasmid pHY300PLK and the recombinant plasmid was transferred into mutant M216. The lecithin-solubilizing ability of mutant M216 was restored. Results suggested that the lecithin-solubilizing ability of NCD-2 was related with phoR gene.Purification and identification of antifungal lipopeptides produced by B. subtilis NCD-2 was also studied in this experiment. Crude lipopeptides were extracted with methanol from the precipitate, which was obtained by adding 6mol/L HC1 to the cell-free culture broth and then stored at 4℃overnight. The crude extract showed antifungal activities against V. dahliae and Botrytis cinerea on the double layer medium. Three mainly peak groups were obtained by HPLC analysis, and only the second compound had inhibitory activity against V. dahliae and B. cinerea. The second compound was analysised by MALDI-TOF MS and two groups of homologues with a difference of 14 Da in molecular weights were identified. In the first group, there were 4 kinds of homologues with the molecular weights of 1446.801,1460.813, 1474.830,1488.852Da, respectively, which were estimated as the isomer of Fengycin A(C15-18). In the second group, the molecular weights of 5 kinds of homologues were 1462.798,1476.817,1490.839,1504.855,1518.867Da, respectively, which were identified as lipopeptide antibiotics Fengycin B(C14-18).