Novel iturin antibiotics of Bacillus subtilis: structure and genetic characterizationSeaim Lwin Aye
( M.Sc. )
A collection of 143 bacterial strains were isolated from the waste of a shrimp factory. One strain showed the highest antimicrobial activity against plant and human pathogens. This strain was identified to be Bacillus subtilis and designated as strain SSE4. HPLC analysis of the bioactive compounds revealed two major peaks that are responsible for the antimicrobial activity. The factors influencing the production of the bioactive compound from B. subtilis SSE4 were determined by varying the culture media and incubation time. Tryptic soy broth (TSB) was found to be the most suitable medium for the production and the activity of bioactive compound. The optimum time for the cultivation was found to be in the range of 48-72 hrs. The structural elucidation of the two bioactive compounds from the major HPLC peaks was performed. It was found that B. subtilis SSE4 produces two antibiotic compounds: iturin A, which is a known antibiotic, and a new member of iturin group which differs from other iturin family members in the presence of a double bond on the side chain of the amino acid chain comprised of 3-amino-13-methyltetradec-8-enoic acid. This new member was designated as subtulene A. The characterization of the genetic operon which is responsible for the iturin antibiotics production showed the presence of three open reading frames as ituD, ituA and ituC. However, the complete set of open reading frames of the iturin operon of B. subtilis SSE4 is under investigation. In order to determine the mechanism of action, and to characterize the target of the iturin and subtulene A antibiotics, a screening of a Tn transposon library of Pseudomonas pudita KT 2440 was performed. A gene responsible for resistance to strain SSE4-derived antibiotics was identified as ompR, which is an osmotic response regulator in P. putida KT 2440. The ompR mutant named IR-1 strain showed resistance towards iturin A and subtulene A. However, the genetic complementation of the ompR Tn-mutant was unsuccessful. The establishment of genetic transformation for the wild type B. subtilis SSE4 was performed using a variety of methods such as natural competency, modified competency, electroporation, protoplasting and combination of electroporation and protoplasing using the plasmid vectors: pMad, pMutin, and pKS. None of the techniques is found to be an efficient method for the transformation of B. subtilis SSE4.
Biocontrol / iturin A / subtulene A / antibiotic / iturin family/ structure elucidation