In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins

Abstract

Sacculus is a peptidoglycan matrix that protects bacteria from osmotic lysis. In Gram-positive organisms, the sacculus is densely functionalized with glycopolymers important for survival, but how assembly occurs is not known. In Staphylococcus aureus, three LCP family members have been implicated in attaching the major glycopolymer wall teichoic acid (WTA) to peptidoglycan, but ligase activity has not been demonstrated for these or any other LCP proteins. Using WTA and peptidoglycan substrates produced chemoenzymatically, we show that all three proteins can transfer WTA precursors to nascent peptidoglycan, establishing that LCP proteins are peptidoglycan-glycopolymer ligases. Although all S. aureus LCP proteins have the capacity to attach WTA to PG, we show that their cellular functions are not redundant. Strains lacking lcpA have phenotypes similar to WTA null strains, indicating that this is the most important WTA ligase. This work provides a foundation for studying how LCP enzymes participate in cell wall assembly.

The cell cycle in Staphylococcus aureus is regulated by an amidase that controls peptidoglycan synthesis

“…7b). When a radiolabel was incorporated into the glycan backbone 20 , we observed a reduction in polymer size but not in the intensity of bands (Fig. 3b and Supplementary Fig.…”

“…Overexpression and purification of S. aureus LcpB: E. coli BL21(DE3) was transformed with plasmid pLcpB encoding S. aureus His 8 -LcpB [S31-N405]. This protein was expressed and purified as previously reported 20 .…”

“…To prepare radiolabeled peptidoglycan oligomers, a [ 14 C]-label was initially incorporated by incubating synthetic Lipid I with UDP-[ 14 C]-GlcNAc (specific activity= 300 nCi/nmol) using MurG as previously published 20 . [ 14 C]-Lipid II was incubated at room temperature with SgtB Y181D (2 µM) in 1X TGase buffer (50 mM HEPES, pH 7.5, and 10 mM CaCl 2 ) in 20% DMSO for 20 min.…”

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A Carbohydrate inStreptococcus pyogenes

“…However, further research will be required to confirm this hypothesis. The last step of GAC biosynthesis is probably similar to the last step of WTA biosynthesis: it involves attachment of GAC to certain MurNAc residues in peptidoglycan via a phosphate ester linkage (46). It is likely catalyzed by members of LytR-CpsA-Psr (LCP) phosphotransferase family encoded by M5005_Spy_1099 and M5005_Spy_1474.…”

High-throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistantStaphylococcus aureusand underlines a tailored response to different osmotic stressors

“…Transcriptome data (Mäder et al, 2016) 188 suggested that gene 957 is in an operon with lcpB ( Fig. 3B), which is one of three wall teichoic acid 189 (WTA) ligases present in S. aureus (Chan et al, 2013, Over et al, 2011, Schaefer et al, 2017. fmtA 190 is located upstream of 957 and has been proposed to code for an esterase that can remove D-191 alanine modifications from teichoic acids (Rahman et al, 2016) and is involved in methicillin 192 resistance (Berger- Bächi et al, 1989).…”