Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase

Abstract

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.

Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli

“…Cellular phospholipids were extracted and analysed as described previously (Okada et al, 1994). The bgalactosidase assay method using ONPG as substrate and the unit definition were as described by Wang & Doi (1984).…”

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis

“…No sequence similarity was identified between the H. pylori PSS and its counterparts from Haemophilus influenzae RD, Mycoplasma pneumoniae, and Mycobacterium genitalium, whose whole genome sequences are now available. The hydropathy profile of the H. pylori PSS protein is also similar to those found within the S. cerevisiae and B. subtilis PSS polypeptides (28,32). The amino acid composition among these four enzymes is primarily hydrophobic.…”

“…PSS activity was assayed with or without the presence of 10 mM MnCl 2 as previously described (32). L- H]serine was purchased from Amersham/Canada (Oakville, Ontario, Canada), whereas phosphatidylserine and CDP-diacylglycerol were obtained from Sigma-Aldrich Canada (Mississauga, Ontario, Canada).…”

“…The E. coli PSS contains 452 amino acids and thus is over 275 amino acids longer than that identified in Bacillus subtilis (6,32). In addition, there is little sequence similarity between these two PSS proteins.…”

“…This feature is consistent with the presence of extensive hydrophilic stretches in its amino acid sequence (6). In contrast, the B. subtilis PSS, like the yeast PSS (28), appears to be a membrane-bound protein (32), as indicated by the fact that the activity of this enzyme in B. subtilis and Bacillus licheniformis is membrane associated (7,32).…”