Isolation and expression of the Rhodobacter sphaeroides gene (pgsA) encoding phosphatidylglycerophosphate synthase

Abstract

The Rhodobacter sphaeroides pgsA gene (pgsA Rs ), encoding phosphatidylglycerophosphate synthase (PgsA Rs ), was cloned, sequenced, and expressed in both R. sphaeroides and Escherichia coli. As in E. coli, pgsA Rs is located immediately downstream of the uvrC gene. Comparison of the deduced amino acid sequences revealed 41% identity and 69% similarity to the pgsA gene of E. coli, with similar homology to the products of the putative pgsA genes of several other bacteria. Comparison of the amino acid sequences of a number of enzymes involved in CDP-diacylglycerol-dependent phosphatidyltransfer identified a highly conserved region also found in PgsA Rs . The pgsA Rs gene carried on multicopy plasmids was expressed in R. sphaeroides under the direction of its own promoter, the R. sphaeroides rrnB promoter, and the E. coli lac promoter, and this resulted in significant overproduction of PgsA Rs activity. Expression of PgsA Rs activity in E. coli occurred only with the E. coli lac promoter. PgsA Rs could functionally replace the E. coli enzyme in both a point mutant and a null mutant of E. coli pgsA. Overexpression of PgsA Rs in either E. coli or R. sphaeroides did not have dramatic effects on the phospholipid composition of the cells, suggesting regulation of the activity of this enzyme in both organisms.Rhodobacter sphaeroides is a gram-negative bacterium capable of growth by aerobic and anaerobic respiration, fermentation, and anoxygenic photosynthesis (34). R. sphaeroides responds to changes in its environment with both physiological and morphological adaptations. Under aerobic conditions, the organism has a typical gram-negative outer membrane and cytoplasmic membrane. At low oxygen tension a dramatic differentiation of the cytoplasmic membrane occurs, with the formation of an intracytoplasmic membrane. The intracytoplasmic membrane is physically continuous with the cytoplasmic membrane but structurally and functionally distinct. The intracytoplasmic membrane contains all of the components necessary for photosynthesis (34). Studies on the regulation of intracytoplasmic membrane assembly have demonstrated the cell cycle-specific insertion of phospholipids into the intracytoplasmic membrane of photoheterotrophically growing cells (13,32), with protein and photopigments incorporated continuously into the intracytoplasmic membrane throughout the cell cycle. Insertion of phospholipids into the intracytoplasmic membrane is concurrent with the onset of cell division and is the result of the net transfer of phospholipids previously synthesized outside the intracytoplasmic membrane (13,32,51). Although the intracytoplasmic membrane and its protein components have been extensively studied, the role specific phospholipid species and general phospholipid metabolism play in the induction, synthesis, assembly, and function of the intracytoplasmic membrane has received only limited attention, mainly because of the lack of detailed biochemical information relating to phospholipid metabolism and lack of genetic information on the...

Isolation and Characterization of the Gene (CLS1 ) Encoding Cardiolipin Synthase in Saccharomyces cerevisiae

“…The yeast CLS1 gene product shows significant homology with the PG-P synthases of prokaryotic organisms (40) and little or no homology with the CL synthase of E. coli (43). Although initially surprising, this result is still consistent with the enzymatic reactions catalyzed by PG-P and CL synthases (51)(52)(53)(54) which are both CDP-DAG-dependent phosphatidyltransferases, whereas the prokaryotic CL synthase does not utilize this liponucleotide.…”

“…This motif is present in the PG-P synthases shown in Fig. 2 and is also found in phosphatidylserine synthases from Bacillus subtilis and S. cerevisiae, in phosphatidylinositol synthase from S. cerevisiae, and in numerous other bacterial PG-P synthases (40); this motif is not found in either the phosphatidylserine synthase (42) or CL synthase (43) …”

A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2‐diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products

“…We detected mainly DGlcD in B. subtilis cultures, whereas in vitro we found MGlcD and DGlcD but no TGlcD. These differences may depend on the actual activity of the enzyme, which may be regulated by the lipid composition of the membrane (Dryden and Dowhan, 1996). A similar autoregulation by lipid environment was described for the synthesis of MGlcD in Acholeplasma laidlawii, in which this effect results in MGlcD homeostasis (Karlsson et al, 1997).…”

Lipidomics for Geochemistry

“…The primary driver of phospholipid biosynthetic diversity is thought to be their multitude of roles in cellular function. PgsA activity first was demonstrated in E. coli (Icho and Raetz, 1983), and pgsA from Rhodobacter is similar to the gene in E.coli (Dryden and Dowhan, 1996). The pathway universally begins with the conversion of phosphatidic acid (PA; diacylglycerol-3-phosphate) to the central intermediate, cytosine diphosphate (CDP)-diacylglycerol (CDP-DAG) (enzyme 38, gene cdsA; Table 1; Figure 7), which then is diverted to its respective fate: PG, PE, or PC.…”