Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across
cisdouble bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1and cfa2,proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1and cfa2mutant strains grown under Pi starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wild-type cells and the cfa1mutant, total cyclopropane fatty acids (CFAs) increased by 10 % and 15 % under Pi starvation and acidic conditions, respectively; whereas in the cfa2mutant, CFAs were less than 0.1 % of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1and cfa2were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2was required for the cyclopropanation of lipids in S. melilotiwhereas the role of cfa1remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under Pi starvation. The cfa2gene was also required for cyclopropanation of non-phosphorus-containing lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1nor cfa2was required for symbiotic nitrogen fixation.