| Bacillus subtilis has long been a model bacterium for understanding biological mechanisms, such as fatty acid catabolism and polyketide biosynthesis. Our interest in the latter was centered on the polyketide synthase (PKS) mechanism responsible for beta-branching polyketides. The unique structural moiety is attributed to a HMG-CoA synthase homolog, such as the pksG gene in B. subtilis..;The first goal was a metagenomic survey of local soils, using the conserved pksG homolog sequence as a genetic marker. After optimizing techniques for the extraction and purification of environmental DNA, the beta-branching polyketide population was not detected in any local soil samples. While working with a pksG homolog, an apparent sequence anomaly prompted us to verify the taxonomic classification of B. subtilis research strains ATCC 39374 and 39320. Comparison of DNA sequences ( pksG homologs, hypervariable regions of 16S rRNA and rDNA) and species-specific genes showed the two ATCC strains are more closely related to B. amyloliquefaciens. .;A group of genes named the mmg operon, able to catalyze fatty acid catabolism, are active only during B. subtilis sporulation. Our hypothesis was that, by creating a conditional genetic knockout mutation, the bacterium would be capable of in-vitro growth in propionate media. Attempts to subclone portions of mmg DNA were unsuccessful. However, the neighboring YqiQ protein was successfully isolated and purified. |
