Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product

Abstract

The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization.The phosphatidyl glycerophosphate (PGP) B phosphatase of Escherichia coli hydrolyzes three substrates: PGP, phosphatidic acid (PA), and lysophosphatidic acid (LPA). Although the membrane localization of PGP phosphatase has not been studied previously, enzymes in the phospholipidbiosynthetic pathway are generally localized in the cytoplasmic membrane in E. coli and Salmonella typhimurium. On the other hand, Bell et al. (2) observed that LPA phosphatase is localized in the outer membrane. Therefore, the finding that the PGP, PA, and LPA phosphatase activities directed by the pgpB gene are not separable genetically (17) raised a question about the substrate specificity and membrane localization of the pgpB gene product. The unusual discovery presented in this paper is that this enzyme is located in both the outer and cytoplasmic membranes. Furthermore, PGP phosphatase activity is higher in the cytoplasmic membrane, whereas PA and LPA phosphatase activities are higher in the outer membrane.To understand this unusual dual subcellular localization, I have cloned this gene and inserted it into an expression vector, a runaway replication plasmid (3) carrying the tac promoter (28), to overproduce this gene product. This study shows that the pgpB gene is a single structural gene coding for an enzyme with all three phosphatase activities. The product of the pgpB gene behaves differently in the cytoplasmic and outer membranes of E. coli. MATERIALS AND METHODSBacterial strains and plasmids. E. coli JM103 (lacJ^) (25) was obtained from New England Biolabs, Beverly, Mass. Strain TI82 (phoA8 pgpB26 recA) was a recA derivative from T174 (17). Other strains are described in Table 1 of the accompanying paper (15).Plasmid pLC26-25 (cysB+ gyrA+ pgpB+) was found among the Clarke and Carbon collection of Col El E. coli hybrid plasmids (6). Plasmids pACYC184, pBR322, and pTI10 were described in the accompanying paper (15). Plasmid pDR540 (28) was from PL Biochemicals, Milwaukee, Wis. Plasmid pMOB45 was described previously (18).An expression vector, pTI5, was constructed by inserting t Present address: Laboratory of Genetics, Department of Biology, The University of Tokyo, Hongo, Tokyo 113, Japa...

Comparative genomics and evolution of eukaryotic phospholipid biosynthesis

“…A separate phospholipid biosynthetic reaction catalyzed by a PAP2 member has been described in bacteria and it is the conversion of PGP to PG catalyzed by the pgpB gene [55,56].…”

The Escherichia coli pgpB Gene Encodes for a Diacylglycerol Pyrophosphate Phosphatase Activity

“…Strain CF30 is a mutant with disrupted alleles in both the pgpA and pgpB genes (25). Plasmid pTI5-217 is a runaway replication plasmid that contains the pgpB gene under the control of a tac promoter (24). Cultures were grown in LB medium and maintained on LB agar.…”

“…A PA phosphatase activity has been identified from the membrane fraction of Escherichia coli (22), and mutants defective in this activity have been isolated that are defined by the pgpB gene (23)(24)(25). The PA phosphatase activity in pgpB mutants has been measured using the assay conditions described for the conventional Mg 2ϩ -dependent PA phosphatase enzyme (23)(24)(25).…”

“…The PA phosphatase activity in pgpB mutants has been measured using the assay conditions described for the conventional Mg 2ϩ -dependent PA phosphatase enzyme (23)(24)(25). However, these assay conditions would not reveal the existence of a Mg 2ϩ -independent PA phosphatase activity such as that displayed by the DGPP phosphatase enzyme.…”

Glycolipozyme membrane protein integrase (MPIase): recent data

“…However, overproduction of dgkA did not affect the DAG expression, suggesting the occurrence of tight regulation as to DAG expression (17). Several genes are known to be involved in DAG biosynthesis, i.e., mdoB (22,23), pgpB (24)(25)(26), and eptB (27). The products of these genes produce DAG as committed reactions.…”