Kennedy pathway for the biosynthesis of E. coli phospholipids. The enzymes responsible for each conversion are named according to their structural genes. In general, a single E. coli gene encodes each enzyme. However, the conversion of PGP into PG is catalyzed by three structurally distinct inner membrane phosphatases. PgpA and PgpB were reported previously (16, 23), but double mutants are viable, accumulate only small amounts of PGP, and synthesize normal levels of PG and CL. The PgpC phosphatase, the subject of this study, accounts for the residual PGP-phosphatase activity. The active site of PgpB faces the periplasm (43), whereas those of PgpA and PgpC likely face the cytoplasm. PS, phosphatidylserine.

High PGP-phosphatase activity in membranes of cells expressing the pgp genes on pBAD33.1. The two known genes, pgpA and pgpB, and the newly identified pgpC gene were cloned into pBAD33.1, and YL7 host cultures were induced with 0.2% (w/v) l-arabinose. A, membranes (0.5 mg/ml) prepared from YL7/pBAD-A, YL7/pBAD-B, and YL7/pBAD-C showed very high levels of phosphatase activity in vitro at 22 °C compared with their vector control membranes. PgpA and PgpC membranes were more active than PgpB membranes under these conditions. B, lipid profiling by LC/MS confirmed that overexpression of pgpA, pgpB, or pgpC greatly reduces the amount of PGP that accumulates in the parental strain YL7, which lacks both pgpA and pgpB. The relative levels of PGP were determined by LC/ESI-MS. They were normalized to the endogenous PE in the same sample, which was defined as 1 for the wild-type W3110, corresponding to about 0.1% of the total lipid (16, 23).

PGP-phosphatase activity in membranes of the double mutants at 22 and 42 °C. PGP-phosphatase activity was measured using 0.5 mg/ml membranes from the wild type or from each of the double mutants with 100 μm carrier PGP. Accordingly, 10% conversion of substrate to product corresponds to 20 nmol/mg protein, which corresponds to a specific activity of ∼9.94 nmol/mg/min for wild-type membranes at 42 °C. A, W3110 membranes are more active at 42 than 22 °C. B, in contrast, membranes of the mutant lacking both PgpA and PgpB are more active at 22 than 42 °C, consistent with previous reports that residual PGP-phosphatase activity (now ascribed to PpgC) is temperature-sensitive in pgpApgpB double mutants (23). C, PgpB is more active at 42 than 22 °C. D, same is true of PgpA. Surprisingly, PgpA activity in YL23 is relatively low at both temperatures. However, only one standard condition was employed for assaying all three phosphatases. The optimal conditions for each enzyme have not yet been determined. Error bars represent the standard deviations of the averages of three determinations.

Temperature-sensitive growth of triple mutant YL24 covered by pMAK705 expressing pgpA, pgpB, or pgpC. A, strains YL24/pMAK-A, YL24/pMAK-B, and YL24/pMAK-C (labeled as A, B, and C, respectively, on the plates) were streaked at 30 or 42 °C. At 42 °C, the complemented strains were unable to form single colonies because of the loss of the covering plasmids. The wild-type W3110 grew well at both 30 and 42 °C. B, same complemented strains were grown on LB broth at 30 °C and then shifted to 42 °C at time 0 (arrow). YL24/pMAK-B grew more slowly than the others at 30 °C. Its overnight culture had reached A₆₀₀ = 0.3 and thus was used directly without additional pre-shift growth at 30 °C. The y axis is the cumulative A₆₀₀ measurement plotted on a log scale, taking into account the back dilutions that were made whenever the cultures reached A₆₀₀ ∼0.2. All three strains stopped growing 3–12 h after the temperature shift.

Expression cloning of the E. coli PgpC phosphatase. Pools of cultures of five E. coli YL7 colonies, harboring different wild-type E. coli DNA fragments on pACYC184, were lysed and assayed for PGP-phosphatase activity at 22 °C using phosphatidyl[U-¹⁴C]glycerol phosphate as the substrate. A, TLC plate used to assess the extent of PGP dephosphorylation was developed in chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v). The 1st two lanes on left are matched extracts of W3110/pACYC184 and YL7/pACYC184, the vector controls. The 2nd lane of the seven representative pools of five colonies shows a significant increase in PGP-phosphatase activity, as judged by the increased intensity of the PG spot (gray arrow). B, PhosphorImager data were quantified and expressed as the % dephosphorylation of the PGP substrate during a 15-min assay. VC, vector control.

Accumulation of PGP in single and double mutants. A, about 2 μg of total lipids, extracted from the indicated strains using the Bligh-Dyer method, were spotted onto a silica TLC plate, which was developed in chloroform/pyridine/formic acid/water (50:50:16:5, v/v). Lipids were detected by sulfuric acid charring. PE is the most abundant lipid in each strain and serves as the loading control. Visible amounts of a band migrating like PGP accumulate in the pgpApgpB and pgpApgpC double mutants. B, LC/MS confirmed the TLC results. YL22 (ΔpgpAΔpgpC) and YL7 (ΔpgpAΔpgpB) accumulated the most PGP, whereas the three single mutants and YL23 (ΔpgpBΔpgpC) did not accumulate very much PGP. The PGP levels were quantified as in Fig. 3.

Mg²⁺ dependence of the three E. coli PGP-phosphatases. The three double mutants analyzed in Fig. 5 were used to assess the Mg²⁺ dependence of the three phosphatases, when assayed using 0.5 mg/ml membranes. The indicated conditions are as follows: Mg, 2 mm MgCl₂ added; E, 2 mm EDTA added; and −, no addition. A no enzyme control was included in which PBS replaced the membranes. The wild-type (WT) membranes contain all three PGP-phosphatases and are therefore not completely inhibited by EDTA. PgpA and PgpC are both Mg²⁺-dependent, whereas PgpB is not.

LC/ESI-MS analysis of YL24/pMAK-A lipids from 30 or 42 °C-grown cells. Total lipids extracted from YL24/pMAK-A were fractionated using normal phase LC and analyzed on line by negative ion ESI-MS (40). A, numbers in the total ion chromatogram of the lipids from 30 °C-grown cells indicate the following peaks: 1, free fatty acids; 2, PG; 3, CL; 4, PE; 5, lyso-PE; and 6, phosphatidic acid and undecaprenyl-phosphate. B, partial spectrum of lipids from 30 °C-grown cells, eluting between 27 and 30 min in the region expected for PGP. No PGP species were detected in this strain at 30 °C. C, numbers in the total ion chromatogram of the lipids from the 42 °C-shifted cells indicate the following peaks: 1, free fatty acids; 2, PG; 3, CL; 3a, lyso-CL; 3b, bis-lyso-CL and methyl PGP; 4, PE; 5, lyso-PE; 6, phosphatidic acid and undecaprenyl-phosphate; and 7, PGP. The lipid composition changed dramatically after the temperature shift with massive accumulation of PGP, loss of PG, and accumulation of lyso-CL, bis-lyso-CL, and methylated PGP. D, partial spectrum of the lipids from the 42 °C-shifted cells eluting between 27 and 30 min in the region expected for PGP. Several strong peaks, corresponding to those expected for molecular species of PGP with the fatty acid composition of E. coli, were present as doubly charged ions. For instance, the peak at m/z 399.21 is attributed to [M − 2H]²⁻ of a PGP species bearing palmitate and palmitoleate. Singly charged ions of the same PGP species were also present at about one-quarter the intensity (not shown). The same lipid analysis is shown for YL24/pMAK-C and YL24/pMAK-B in supplemental Figs. 1 and 2 with similar conclusions.