Volume 23, Issue 5, Pages 765-772 (September 2006) Acyl-Phosphates Initiate Membrane Phospholipid Synthesis in Gram-Positive Pathogens  Ying-Jie Lu, Yong-Mei Zhang, Kimberly D. Grimes, Jianjun Qi, Richard E. Lee, Charles O. Rock  Molecular Cell  Volume 23, Issue 5, Pages 765-772 (September 2006) DOI: 10.1016/j.molcel.2006.06.030 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 PlsX Is a Phosphate:acyl-ACP Acyltransferase (A) Purified SpPlsX was a soluble protein with a Stoke's radius determined by gel filtration chromatography consistent with its existence as a dimer in solution. The insets show the calibration of the column and an SDS gel of the purified SpPlsX preparation. (B) The activity requirements for the formation of acyl-PO4 from [14C]16:0-ACP by SpPlsX were assessed by thin-layer chromatography of the reaction products on Silica Gel G layers developed with chloroform:methanol:acetic acid (90/10/10). (C) Acyl-ACP, but not fatty acid or 16:0-CoA, was a substrate for SpPlsX, which also incorporated [32P]orthophosphate into the product as determined by thin-layer chromatography on Silica Gel G layers developed with chloroform:methanol:acetic acid (90/10/10). The asterisk (∗) indicates the radiolabeled substrate used in the assay. (D) Negative ion electrospray precursor ion scanning MS of the product of the SpPlsX reaction using 16:0-ACP and phosphate. (E) The PlsX reaction was reversible, catalyzing the synthesis of acyl-ACP from 16:0-PO4 as determined by conformationally sensitive gel electrophoresis and staining with Coomassie blue. (F and G) The apparent SpPlsX KM values for acyl-ACP (F) and orthophosphate (G) were 300 μM and 140 μM, respectively. The data shown are the means of three independent experiments with the standard errors. Molecular Cell 2006 23, 765-772DOI: (10.1016/j.molcel.2006.06.030) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Acyl-PO4-Dependent G3P Acyltransferase Activity in Bacteria (A and B) Formation of acylated G3P in membrane preparations from S. pneumoniae (A) or E. coli (B) in the presence of 100 μM [14C]G3P and either 16:0-PO4 (•), 16:0-ACP (○), or 16:0-CoA (□) as the acyl donor using the filter disc assay. The data shown are the means of three independent experiments with the standard errors. (C and D) Thin-layer chromatography identification of the acylated products of the reaction of S. pneumoniae (C) or E. coli (D) membranes with 100 μM [14C]G3P with different combinations of acyl donors using Silica Gel H layers developed with chloroform:methanol:acetic acid:water (50/25/8/2). Molecular Cell 2006 23, 765-772DOI: (10.1016/j.molcel.2006.06.030) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 PlsY Is an Acyl-PO4-Dependent G3P Acyltransferase (A) 16:0-PO4 G3P acyltransferase activity in membranes prepared from E. coli strain SJ361 (plsB26 plsX50 plsC1[Ts]) harboring either an empty vector (pPJ131, □) or a plasmid directing the production of SpPlsY (pPJY, ▪), and E. coli strain FB23281 (ygiH::Tn5) with the E. coli plsY homolog inactivated by insertion of the Tn5 element (•) using the filter disc assay. Acyl-G3P was the only product detected, and the inset shows the acyl-G3P region of a thin-layer chromatogram of the products of the reactions using 4 μg of membrane protein. The data shown are the means of three independent experiments with the standard errors. (B) Purified SpPlsY was assayed for G3P acyltransferase activity in the presence of 100 μM [14C]G3P and either 16:0-PO4 (•), 16:0-ACP (○), or 16:0-CoA (□). Acyl-G3P was the only product detected by thin-layer chromatography (data not shown). The inset shows an SDS gel of purified SpPlsY. SpPlsY has a predicted molecular weight of 23 kDa but migrates as a 19 kDa protein in SDS gel electrophoresis. The data shown are the means of three independent experiments with the standard errors. (C) Position of the acyl chain in the PlsY reaction product. Acyl-glycerol was prepared as described in the methods section. The location of the labeled monoacylglycerol (1-MG, Rf = 0.16) was visualized using a PhosphoImager screen. Standards were commercial 1-MG (Rf = 0.16) and 2-monoacylglycerol (2-MG, Rf = 0.38) prepared by the action of Rhizomucor miehei lipase on diacylglycerol. The only labeled species detected in the reactions was the 1-acyl isomer. (D) Negative ion electrospray precursor ion scanning MS of acyl-phosphates isolated from E. coli strain FB23281 harboring plasmid pBlue-spPlsX. (E) Product formation in the presence of a mixture of 16:0-PO4 plus 16:0-ACP. Membranes were prepared from strain SJ361 (plsB26 plsX50 plsC1[Ts]) harboring either an empty expression vector (pPJ131), a SpPlsY expression vector (pPJY), or a SpPlsC expression vector (pYL12). Molecular Cell 2006 23, 765-772DOI: (10.1016/j.molcel.2006.06.030) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 The PlsX/Y Pathway for PtdOH Formation Is the Predominant Route to PtdOH in Bacteria (A) The most widely distributed pathway starts with the conversion of a long-chain acyl-ACP end product of fatty acid synthesis to an acyl-PO4 by PlsX. PlsY transfers the fatty acid from the acyl-PO4 to G3P. Acyl-G3P is then converted to PtdOH by PlsC using acyl-ACP as the acyl donor. (B) The alternate pathway proceeds by the PlsB-catalyzed transfer of a fatty acid to G3P from acyl-ACP (or acyl-CoA) followed by the acylation of acyl-G3P by PlsC. (C) Distribution of the PlsB (red), PlsX/Y (green), and PlsC (blue) acyltransferase genes in bacteria. PlsX/Y occurred in 314 genomes, PlsC occurred in 350 genomes, and PlsB occurred in 102 genomes in the Integrated Microbial Genomes database (a total of 271 finished/124 draft sequences were interrogated). Molecular Cell 2006 23, 765-772DOI: (10.1016/j.molcel.2006.06.030) Copyright © 2006 Elsevier Inc. Terms and Conditions