Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

Abstract

Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group acrosscisdouble bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes,cfa1andcfa2,proposed to code for CFA synthases inSinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type andcfa1andcfa2mutant strains grown under Pistarvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wild-type cells and thecfa1mutant, total cyclopropane fatty acids (CFAs) increased by 10 % and 15 % under Pistarvation and acidic conditions, respectively; whereas in thecfa2mutant, CFAs were less than 0.1 % of wild-type under both growth conditions. Reporter gene fusion experiments revealed thatcfa1andcfa2were expressed at similar levels in free-living cells. Thus under the conditions we examined,cfa2was required for the cyclopropanation of lipids inS. melilotiwhereas the role ofcfa1remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred oncis-11-octadecenoic acid located in either thesn-1 or thesn-2 position in phospholipids and that cyclopropanation in thesn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under Pistarvation. Thecfa2gene 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. Neithercfa1norcfa2was required for symbiotic nitrogen fixation.