A widespread family of bacterial cell wall assembly proteins

Abstract

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall.

Figure 1

Lethal phenotype of a triple mutant of mreB paralogues and tagTUV, and rescue by disruption of tagO. (A) Growth of strains 168 (wild-type), YK992 (ΔtagO), YK1190 (amyE∷P_spacHY-mbl ΔmreB Δmbl ΔmreBH ΔtagO), and YK1119 (amyE∷P_spacHY-mbl ΔmreB Δmbl ΔmreBH) on NA plates with or without 0.1 mM IPTG. (B–E) Cell morphologies of typical fields of strains 168 (wild-type, B), RE204 (ΔtagTU, C), YK915 (ΔtagUV, D), and YK917 (ΔtagTV, E). The cell membranes were stained with Nile Red. Scale bar represents 5 μm. (F–H) Cell morphologies of typical fields of strain YK914 (ΔtagTU P_spac-tagV) cultured in the presence (F) or absence (G, H) of 0.5 mM IPTG. Images were taken at 180 (G) and 240 (H) min after removal of IPTG. The cell membranes were stained with Nile Red. Scale bar represents 5 μm. (I) Growth of strains YK1031 (ΔtagTU P_spac-tagV pMAP65), YK1030 (ΔtagTU P_spac-tagV ΔtagO), YK1033 (ΔtagTUV ΔtagO), and YK1163 (ΔtagO) on LB plates with or without 0.5 mM IPTG. (J, K) Cell morphologies of typical fields of strains YK1163 (ΔtagO, J) and YK1033 (ΔtagTUV ΔtagO, K). The cell membranes were stained with Nile Red (right). Scale bar represents 5 μm.

Figure 2

The WTA biosynthetic machinery and LCP family of proteins associated with the MreB cytoskeleton. (A) Summary of proteins associated with MreB cytoskeleton (see also full data in Supplementary Table SI). WTA synthetic and LCP proteins are indicated in blue and red, respectively. (B) Genetic organization of three AP systems in B. subtilis, the WTA biosynthetic genes (blue), the teichuronic acid (TU) biosynthetic genes (green), and the minor TA biosynthetic genes (yellow). lytR homologues, tagT, U, and V, were indicated in red. Numbers show the position on the B. subtilis chromosome.

Figure 3

Effects of tagTUV mutants on WTA synthesis or assembly. (A, B) Cells of 168 (wild-type, lanes 1 and 6), RE201 (ΔtagU, lane 2), YK914 (ΔtagTU P_spac-tagV, lanes 3 and 4), and YK992 (ΔtagO, lane 5) were cultured with (lane 3) or without (lanes 1, 2 and 4–6) IPTG. Purified WTA samples were separated and visualized as described in the Materials and methods (A). Phosphate content of cell wall was assayed as described in the Materials and methods (B). White numbers indicate standard deviation from three independent experiments.

Figure 4

The structure of ΔTM-Cps2A. (A, B) Orthogonal views of the extracellular portion of Cps2A, shown as a cartoon, with the accessory domain coloured red and the LCP domain coloured green. The decaprenyl-phosphate present in the active site is shown as a stick model, with carbon atoms coloured yellow, phosphorous in orange, and oxygen in red. Secondary structure elements in both domains are labelled from N- to C-termini of each domain.

Figure 5

Lipid binding by the LCP domain of ΔTM-Cps2A. (A, B) decaprenyl-phosphate binding to ΔTM-Cps2A and (C, D) octaprenyl-pyrophosphate bound ΔTM-Cps2A. Protein residues within 4 Å of the lipid are shown as stick models, with final, σ^A-type 2mF_obs–DF_calc electron density maps shown as blue transparent surfaces and contoured in both cases at 1.0 σ.

Figure 6

The biochemical properties of LCP proteins. (A) Growth of strains YK1254 (wild-type GFP-TagU), YK1365 (D75A), YK1366 (R83A), YK1367 (D85A), YK1368 (T86F), and YK1369 (T197F) on NA plates with 0.5 mM IPTG or 0.5% xylose. Western blot analysis of GFP-TagU levels in strain expressing wild-type and various mutant GFP-TagU fusions with or without (No) 0.5% xylose indicate correct accumulation of the proteins and consequently mutations D75A, R83A, D85A, T86F, and T197F in TagU are unable to sustain growth in the absence of wild-type copy of tagT, tagU, and tagV. (B) Metal binding by ΔTM-Cps2A. Crystals of Cps2A soaked in manganese show a clear peak in an anomalous difference map calculated for data collected at the manganese K-edge (orange mesh). Metal coordinating residues are shown as ball and stick representations, with the position of R267A shown with blue carbons. (C) Incubation of ΔTM-CPS2A–octaprenyl-pyrophosphate complex with magnesium releases inorganic phosphate. Addition of EDTA, or mutation to alanine of aspartate 234, which coordinates the catalytic magnesium ion, both suppress release of inorganic phosphate. Bar length is proportional to the wild-type experiment with Mg²⁺ present (76.31±2.9 μM released with Mg²⁺ present versus 20.60±1.26 μM released with EDTA present; 3.38±0.68 released by D234A). (D) Pyrophosphatase activity of ΔTM-TagT. A purified ΔTM-TagT protein catalyses hydrolysis of the pyrophosphate bond in exogenously added geranyl pyrophosphate (ger-PP) to generate geranyl monophosphate (ger-P), monitored by thin layer chromatography. Parallel incubations with the unrelated protein, EzrA (purified using the same procedures as the LCP proteins herein) and ΔTM-Cps2A reveal no pyrophosphatase activity towards exogenous ger-PP; in the latter case, this can be attributed to the blockage of the active site by endogenous opr-PP lipid. Hydrolysis is magnesium dependent, but is not affected by the addition of the PG fragment, N-acetylmuramyl dipeptide (MDP). (E) Electrostatic protein surface of the LCP domain of ΔTM-Cps2A, with bound octaprenyl-pyrophosphate ligand. The scissile bond in the phosphotransferase reaction catalysed by this enzyme class is shown with an arrow. The potential pathway for AP binding is highlighted in blue, semi-transparent rod (labelled AP), and the likely position of PG, to which the phosphorylated AP will be transferred, is shown with the green rod (labelled PG).