PgpP is a broadly conserved phosphatase required for phosphatidylglycerol lipid synthesis

Abstract

The cytoplasmic membrane of bacteria is composed of a phospholipid bilayer made up of a diverse set of lipids. Phosphatidylglycerol (PG) is one of the principal constituents and its production is essential for growth in many bacteria. All the enzymes required for PG biogenesis in Escherichia coli have been identified and characterized decades ago. However, it has remained poorly understood how gram-positive bacteria perform the terminal step in the pathway that produces this essential lipid. In E. coli, this reaction is mediated by three functionally redundant phosphatases that convert phosphatidylglycerophosphate (PGP) into PG. Here, we show that homologs of these enzymes in Bacillus subtilis are not required for PG synthesis. Instead, we identified a previously uncharacterized B. subtilis protein, YqeG (renamed PgpP), as an essential enzyme required for the conversion of PGP into PG. Expression of B. subtilis or Staphylococcus aureus PgpP in E. coli lacking all three Pgp enzymes supported the growth of the strain. Furthermore, depletion of PgpP in B. subtilis led to growth arrest, reduced membrane lipid staining, and accumulation of PGP. PgpP is broadly conserved among Firmicutes and Cyanobacteria. Homologs are also present in yeast mitochondria and plant chloroplasts, suggesting that this widely distributed enzyme has an ancient origin. Finally, evidence suggests that PgpP homologs are essential in many gram-positive pathogens and thus the enzyme represents an attractive target for antibiotic development.

Keywords: Bacillus subtilis; PGP phosphatase; phosphatidylglycerol; phospholipid synthesis.

Fig. 1.

B. subtilis strains lacking homologs of E. coli PgpA and PgpB are viable. (A) Schematic of the PG and LTA biosynthesis pathways in B. subtilis. Enzymes with known functions are shown in blue. The PGP phosphatase, PgpP (formally, YqeG) that catalyzes the terminal step in PG synthesis and identified in this study is shown in orange. The flippase(s) required to transport PG and DAG across the membrane (shown in gray) are unknown. (B) Growth curves of WT and the indicated single, double, triple, and quadruple mutants. (C) Photograph of spot-dilutions of the indicated strains on LB agar plates. The growth curve and spot-dilutions are representative results from one of four biological replicates.

Fig. 2.

The S. aureus and B. subtilis pgpP gene can rescue an E. coli pgp depletion strain. (A) Schematic of the genomic loci containing pgpP in S. aureus and B. subtilis. The operons containing pgpP (formally, yqeG) are depicted with genes drawn approximately to scale and gene names and protein function indicated. (B) Spot dilutions of E. coli strain YL24 pMAK-C containing the EV pCB095 or derivatives with E. coli pgcA_EC or pgpC_EC, S. aureus pgpP_SA or B. subtilis pgpP_BS. Strains were serially diluted and spotted onto an LB agar plate containing arabinose and incubated at 30 °C (permissive condition) or LB agar containing xylose and incubated at 42 °C (nonpermissive condition). At 42 °C bacteria can only grow if a functional PGP phosphatase is supplied on the pCB095 plasmid. Representative images from five independent experiments are shown.

Fig. 3.

PgpP is essential in B. subtilis and its depletion leads to membrane defects. (A) Schematic of the IPTG-inducible pgpP (i-pgpP) strain. (B) Growth curves of the indicated strains in the presence and absence of IPTG. The average reading and SD from three experiments were plotted. (C) Schematic of the IPTG-inducible pgpP (i-pgpP) strain with xylose-inducible complementation alleles. (D) Spot dilutions of the i-pgpP complementation strains constructed with the EV or with xylose-inducible E. coli pgcA_EC or pgpC_EC, S. aureus pgpP_SA or B. subtilis pgpP_BS. Strains were serially diluted and spotted onto LB agar plates containing 250 µM IPTG (Left) or 20 mM xylose (Right). Representative images from three independent experiments are shown. (E) Representative phase-contrast and fluorescence images of WT B. subtilis and the i-pgpP strain. Cultures grown for 3 h in the presence or absence of IPTG were stained with PI and the membrane dye TMA-DPH and analyzed by phase-contrast and fluorescence microscopy. The PI fluorescence and phase-contrast images were overlaid to better visualize PI-positive cells (Phase + PI). TMA-DPH fluorescence (membrane) was false-colored yellow. The exposure time and scaling for all four PI and TMA-DPA images were identical. Representative images from one of three independent experiments are shown.

Fig. 4.

The predicted structures of B. subtilis PgpP and the yeast mitochondrial PGP phosphatase Gep4 are similar and their active site aspartic acids are essential for function. (A) AlphaFold models of Gep4 and B. subtilis PgpP. Left panel: Overlay of the Gep4 (AF-P38812-F1-model_v4) (purple) and B. subtilis PgpP (AF-P52254-F1-model_v4) (cyan). Middle and Right panels: Surface representations of Gep4 and PgpP models with the active site aspartic acid residues highlighted in pink. The images were generated with PyMOL v 2.5.3. (B) Spot dilutions of the i-pgpP complementation strain constructed with an EV or with pgpP, pgpP-His, or the active site variants pgpP_D34N-His or pgpP_D36N-His expressed from a xylose-inducible promoter. Strains were serially diluted and spotted onto LB agar plates containing 250 µM IPTG (Left) or 20 mM xylose (Right). Representative images from one of three independent experiments are shown. (C) Immunoblot analysis of PgpP-His or active site variants detected with an anti-His-tag antibody. Data from all strains analyzed are shown in SI Appendix, Fig. S5 along with the SigA protein loading controls. A representative immunoblot from one of three biological replicates is shown.

Fig. 5.

Depletion of PgpP causes an accumulation of PGP in B. subtilis. (A) TLC of total lipids extracted from WT B. subtilis and the PgpP depletion strain (i-pgpP) following growth in LB medium with or without IPTG. The lipids were separated by TLC using an ethylacetate:2-propanol:ethanol:5% ammonia solvent system as described by Osman et al. (30), stained with primulin and the plate imaged. A representative image from three independent experiments is shown. (B) MS traces of PG and PGP. Total membrane lipids isolated from WT or the PgpP depletion strain (i-pgpP) grown in LB without IPTG were analyzed by MS in the positive ion mode. Traces for the extracted masses corresponding to PG and PGP lipids with a combined fatty acid carbon chain length of 30:0, 31:0, 32:0 are shown. One representative set of traces from three independent experiments is shown. The complete extracted mass traces are shown in SI Appendix, Fig. S8, including traces for lipids isolated from the strains grown in the presence of IPTG. (C) Bar graph of the ratios of PGP/PG in the indicated strains and growth conditions. The area of all PG and PGP peaks as shown in (B) and SI Appendix, Fig. S8 were integrated, and the average and SD of the PGP/PG ratios from three independent samples were plotted.

Fig. 6.

PgpP is broadly conserved in Firmicutes, Cyanobacteria, Mollicutes, Deinococcus-Thermus, and Thermotogae. Phylogenetic tree showing the distribution of PgsA, PgpA, and PgpP homologs in Bacteria and Archaea. The phylogenetic tree was constructed using the Phylo T v2 web site (http://phylot.biobyte.de) and the 1,187 bacterial and 122 archaeal taxa included in the COG database (33). The taxa in which PgsA (COG0558, shown in red), PgpA (COG1267, shown in blue), and PpgP (YqeG) (COG02179; shown in orange) proteins are found were mapped onto the tree using iTOL V6 (34). Branches with some of the main phyla and classes are indicated on the perimeter of the tree. Phyla and classes in which PgsA homologs are largely absent are shown in red and phyla and classes in which PgpA and PgpP are both absent are shown in gray. PgpP homologs, shown in orange, are found in Firmicutes, Cyanobacteria, Mollicutes, Deinococcus-Thermus, and Thermotogae as well as the class of Coriobacteriia within the Actinobacterium Phylum.