UNLABELLED

Bacillus subtilis Bs-916 has obvious effects against Rhizoctonia solani. We demonstrated that the mutant strain, M49 obtained by means of low energy ion implantation in strain Bs-916, which produces significantly lower levels of surfactin, had no obvious effects against R. solani.

OBJECTIVE

In order to identify the influencing factors of surfactin-decrease mutant strain M49, its phenotype and related gene expression levels were studied.

METHODS

Strains to be tested for sporulation were grown for 24 h in sporulation medium. Plasmid pDG1728 (1 microg/mL) was used for DNA transformation to test competence development of M49 and Bs-916 strains. RT-PCR (Semi-quantitative reverse transcriptase-polymerase chain-reaction) was used to determine the expression levels of selected genes involved in competence and sporulation in both the wildtype Bs-916 and mutant M49 strains, such as comS, rapA, rapC gene.

RESULTS

Our data showed that mutant strain M49 confers a leaky competence phenotype, typified by a ten-fold reduction. This indicated a fact that DNA fragments are more easily transformed to wildtype strain than M49 mutant. The M49 strain also appeared to exhibit a sporulation-deficient phenotype, compared with the wild-type Bs-916, its spore's number declined by about 75%. RT-PCR results showed that both the comS and rapC genes were expressed in the Bs-916 strain but not in the M49 strain.

CONCLUSIONS

Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter, PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild type Bs-916 and mutant M49 strains is significantly different. When we integrated comA orf into the chromosome of M49 at amyE locus, M49 restored antifungal activity. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis.