CLONING, SEQUENCING, AND DISRUPTION OF THE BACILLUS-SUBTILIS PSD GENECODING FOR PHOSPHATIDYLSERINE DECARBOXYLASE

The psd gene of Bacillus subtilis Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced, It encodes a polypeptid e of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream of pss, the structural gene for phosphatidy lserine synthase that catalyzes the preceding reaction in phosphatidyl ethanolamine synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Mat sumoto, J. Bacteriol. 176:7456-7461, 1994). Introduction of a plasmid containing the psd gene into temperature-sensitive Escherichia coli ps d-2 mutant cells allowed growth at otherwise restrictive temperature, Phosphatidylserine was not detected in the psd-2 mutant cells harborin g the plasmid; it accumulated ire the mutant pip to 29% of the total p hospholipids without the plasmid, An enzyme activity that catalyzes de carboxylation of C-14-labeled phosphatidylserine to form phosphatidyle thanolamine was detected in E. coli psd-2 cells harboring a Bacillus p sd plasmid. E. coli cells harboring the psd plasmid, the expression of which was under the control of the T7 phi 10 promoter, produced prote ins of 32 and 29 kDa upon induction, A pulse-labeling experiment sugge sted that the 32-kDa protein is the primary translation product and is processed into the 29-kDa protein. The psd gene, together with pss, w as located by Southern hybridization to the 238- to 306-k6 SfiI-NotI f ragment of the chromosome, A B. subtilis strain harboring an interrupt ed psn allele, psd1::neo, was constructed, The null psd mutant contain ed no phosphatidylethanolamine and accumulated phosphatidylserine, It grew well without supplementation of divalent cations which are essent ial for the E. coli pssA null mutant lacking phosphatidylethanolamine. In both the B. subtilis null pss and psd mutants, glucosyldiacylglyce rol content increased two- to fourfold. The results suggest that the l ack of phosphatidylethanolamine in the B. subtilis membrane mag be com pensated for by the increases in the contents of glucosyldiacylglycero ls by an unknown mechanism.