Cloning, Sequencing, and Disruption of theBacillus subtilis psd Gene Coding for Phosphatidylserine Decarboxylase

摘要

The psd gene of Bacillus subtilis Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced. It encodes a polypeptide of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream ofpss, the structural gene for phosphatidylserine synthase that catalyzes the preceding reaction in phosphatidylethanolamine synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Matsumoto, J. Bacteriol. 176:7456–7461, 1994). Introduction of a plasmid containing the psd gene into temperature-sensitiveEscherichia coli psd-2 mutant cells allowed growth at otherwise restrictive temperature. Phosphatidylserine was not detected in the psd-2 mutant cells harboring the plasmid; it accumulated in the mutant up to 29% of the total phospholipids without the plasmid. An enzyme activity that catalyzes decarboxylation of14C-labeled phosphatidylserine to form phosphatidylethanolamine was detected in E. coli psd-2cells harboring a Bacillus psd plasmid. E. colicells harboring the psd plasmid, the expression of which was under the control of the T7φ10 promoter, produced proteins of 32 and 29 kDa upon induction. A pulse-labeling experiment suggested that the 32-kDa protein is the primary translation product and is processed into the 29-kDa protein. The psd gene, together withpss, was located by Southern hybridization to the 238- to 306-kb SfiI-NotI fragment of the chromosome. AB. subtilis strain harboring an interrupted psdallele, psd1::neo, was constructed. The null psd mutant contained no phosphatidylethanolamine and accumulated phosphatidylserine. It grew well without supplementation of divalent cations which are essential for theE. coli pssA null mutant lacking phosphatidylethanolamine. In both the B. subtilis null pss andpsd mutants, glucosyldiacylglycerol content increased two- to fourfold. The results suggest that the lack of phosphatidylethanolamine in the B. subtilis membrane may be compensated for by the increases in the contents of glucosyldiacylglycerols by an unknown mechanism.