Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway.

A characteristic among many marine Gammaproteobacteria is the bio-synthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of eicosapentaenoic acid (EPA) and/or docosa-hexaenoic acid (DHA) is mediated by a polyketide/fatty acid synthase mechanism encoded by a set of five genes, pfaABCDE. This unique fatty acid synthesis pathway coexists with the principal type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of core saturated, monounsaturated, and hydroxy-lated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a ge-netic approach was undertaken to elucidate genetic regulation of the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon is downregulated in response to exogenous fatty acids, particularly long-chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present in P. profundum SS9. Transposon mutagenesis and screening of a li-brary of mutants identified a novel transcriptional regulator, which we have desig-nated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site. IMPORTANCE The production of long-chain omega-3 polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environ-ments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their prod-ucts, and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this path-way is regulated under various culture conditions and has identified and character-ized a fatty acid-responsive transcriptional regulator specific to PUFA biosynthesis.