Membrane lipids analysis of a bacterial strain 17560 isolated from nodule of Alfalfa and cloning and expression of the genes for diacylglyceryl trimethylhomoserine (DGTS) biosynthesis

Abstract:

[Objective] The aim of this study is to analyse membrane lipids composition of a rhizobial strain isolated from nodule of Alfalfa in Heilongjiang province under phosphorus-limited condition and to clone and identify the genes required for diacylglyceryl trimethylhomoserine (DGTS) biosynthesis. [Methods] The rhizobial strain was cultured in Sherwood minimal medium containing either normal or low concentrations of inorganic phosphate.The membrane lipids were extracted by Bligh-Dyer method and were analysed by thin-layer chromatography (TLC) using both the lipids of Sinorhizobium meliloti 1021 and standard samples of some phospholipid as a reference. PCR primers was designed according to the sequences of btaA and btaB in the GenBank. The PCR-amplified btaA and btaB genes were expressed in Escherichia coli BL21(DE3). The gene function was verified by testing DGTS production. 16S rRNA gene sequences of the strain 17560 was analysed. [Results] The strain 17560 isolated from Alfalfa in Heilongjiang province shares 99.8% 16S rRNA gene sequence identity with Sinorhizobium meliloti. But its lipid compositions were quite different from that of the reference strain S. meliloti 1021. Under phosphorus-limited conditions, the main membrane lipids of the strain17560 were phosphorus-free lipids, such as ornithine lipid (OL) and DGTS. Strain 17560 contain three different types of OLs meanwhile S. meliloti 1021 contain only one type of OL. Under normal phosphorus condition, the main membrane lipids of strain 17560 were phospholipid, such as phosphatidylethanolamine (PE) and one unkonw amino-containing phospholipid, while the main membrane lipids of S. meliloti 1021 are PE, phosphatidylcholine (PC) and phosphatidylglycerol (PG). BtaA and btaB genes of strain 17560 were amplified. One PCR product of 1 913 bp was obtained, which contains two ORFs sharing 99% sequence identity with btaA and btaB genes of S. meliloti 1021 respectively. This DNA fragment was cloned to pET-30a(+) vector and transferred to E. coli BL21(DE3). The proteins of 45 kD and 25 kD were detected, and TLC analysis further revealed DGTS production in the expression strain when induced with isopropyl-b-D-thiogalactoside (IPTG). [Conclusion] Membrane lipids of rhizobial strains from Alfalfa could be quite different although their phylogenetic position were the same. Membrane lipids of rhizobial strain varies with the phosphorus content in the growth medium, the main membrane lipids of the strain were phosphorus-free lipids, such as OL and DGTS under phosphorus-limiting condition. One unknown amino-containing phospholipid and three different types of OLs was found in Sinorhizobium. The btaA and btaB genes were cloned and expressed successfully in E. coli.

Key words:Rhizobia from Alfalfa, Phosphorus-free membrane lipids, Diacylglyceryl trimethylho-moserine, Thin-layer chromatography (TLC), Cloning and expression of btaA and btaB genes