A similar picture was seen for the FabF proteins, one (now called FabO) performed the FabB function whereas the other functioned only as a FabF [9]. However, neither of these scenarios seemed applicable to the Clostridia. C. acetobutylicium lacks fabM, fabA and

fabB and has only a single copy of fabZ, although its fatty acid composition is similar to that of E. coli. This bacterium contains three genes that encode putative FabFs, although only one of these seemed likely to be involved in fatty acid synthesis (see Discussion). The most likely LY2603618 FabF homologue candidate was that encoded within a large gene cluster (fabH acpP fabK, fabD fabG fabF accB fabZ accC accD accA) that encodes what appears to be a Romidepsin datasheet complete set of the genes

required for saturated fatty acid synthesis. How does C. acetobutylicium make unsaturated fatty acids? One possibility was that the single FabZ and FabF homologues could somehow function in both the saturated and unsaturated branches of the fatty acid synthetic pathway. We report that the C. acetobutylicium FabZ cannot catalyze isomerization of its trans-2-decenoyl-ACP product to the cis-3 species either in vitro or when expressed in E. coli. However, the single FabF homologue active in fatty acid synthesis has the functions of both E. coli long chain 3-ketoacyl-ACP synthases, FabB and FabF. Figure 1 Unsaturated fatty acid biosynthetic pathway of E. coli. Results Only one of the three C. acetobutylicium fabF homologues can functionally replace E. coli FabF in vivo There are three annotated C. acetobutylicium fabF homologues designated as CAC3573, CAC2008

and CAA0093 [10]. We will temporarily call these genes fabF1, fabF2 and fabF3, although our data indicate that only the first of these genes functions in fatty acid synthesis. To test the functions of these homologues, the three genes were inserted into the arabinose-inducible vector pBAD24. The resulting plasmids were then introduced into two E. coli fabB(Ts) fabF strains, CY244 and JWC275. At the non-permissive temperature these mutant Foretinib clinical trial strains lack both long chain 3-ketoacyl-ACP synthase activities and thus are unable to grow even when the medium is supplemented with the unsaturated fatty acid, oleate [11, 12]. Derivatives of strains CY244 or JWC275 carrying pHW36 encoding fabF1 grew at 42°C in the presence of oleate whereas the strains STK38 carrying pHW37 and pHW38 (encoding fabF2 and fabF3, respectively) failed to grow (Fig. 2) (similar results were seen with plasmids of both low and high copy number vectors). Thus, only fabF1 complemented the E. coli fabF mutation showing that C. acetobutylicium FabF1, like E. coli FabF, is able to catalyze all of the elongation reactions required in the synthesis of saturated fatty acids. Furthermore, expression of FabF1 restored thermal control of fatty acid composition to a FabF null mutant strain (Table 1). An E. coli fabF strain in which C.