Study Of The Lipopeptide Antibiotics Iturins From Bacillus Thuringiensis

Many different Bacillus spp., especially Bacillus subtilis strains often produce a variety of antimicrobial cyclic lipopeptides, including the non-ribosomal biosynthesis of surfactins, fengycins, and iturins. The iturins shows a strong antibiotic activity with a broad antifungal spectrum, making it an ideal potential biological control agent with the aim of reducing the use of chemical pesticides in agriculture and medical science. This study used three methods, such as polymerase chain reaction (PCR) , fungal growth inhibition assay and quantitative high-performance liquid chromatography (HPLC) to identify whether a number of Bacillus strains can produce iturin A. In order to find the iturins in the B. thuringiensis which has closer genetic relationship with B.subtilis but had not been found to produce iturins.This study made use of the Bacillus thuringiensis, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus to examine the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD amplification. Nine of Bacillus strains exhibited positive results. Blast sequence analysis shows that the PCR products are over 95% homologous to genes of iturins operon. Crude lipopeptides were extracted with methanol from the precipitate which were obtained by adding 6 mol/L HC1 to the cell-free culture broth. The assay for potential production of iturin A was carried out by placing crude lipopeptides of the tested Bacillus strains on a PDA agar plate with Colletotrichum musae, Fusarium oxysporum, Botrytis cinerea and Rhizoctonia solani. The fungal growth inhibition assay exhibited that four Bacillus strains-- Bacillus subtilis EPL8, Bacillus subtilis BS1, Bacillus amyloliquefaciens TB2, Bacillus thuringiensis BRC-XQ16 have inhibitory activity against some important plant pathogenic fungi.Crude lipopeptides of the four antifungal Bacillus species were purified and then run on lichrosphere C18 column in reverse phase HPLC system. With HPLC method , it was determined that the antifungal lipopeptides was composed of many components , so as the iturinA standard, because it has different homologues . Through the HPLC analysis , it shows that three major components of the lipopeptides extracted from EPL8 and TB2 had similar retention time to three components of the iturinA standard. The results indicates that the three major components of the lipopeptides extracted from EPL8 and TB2 maybe the other iturin family members , such as mycosubtilin and bacillomycin D. The major component of the lipopeptides extracted from BS1 has almost the same retention time as the major component of iturinA standard, and it maybe just the major component of the iturinA standard. Two major components of the lipopeptides extracted from BRC-XQ16 had similar retention time to two components of the iturinA standard. As the analysis above, the results indicated that the two major components of the lipopeptides extracted from BRC-XQ16 maybe the other iturin family members , such as mycosubtilin and bacillomycin D.Based on the above analysis , the four Bacillus strains , Bacillus subtilis EPL8, Bacillus subtilis BS1, Bacillus amyloliquefaciens TB2, Bacillus thuringiensis BRC-XQ16 could product the iturinA or other iturin family members.The result of this study settle the basis for the further study on the determination of structures of the lipopeptides extracted from EPL8, TB2, BS1 and BRC-XQ16. If the speculation concluded from the HPLC were consistent with the result of the determination of structures,and widen the application of the B. thuringiensis.