The Cpx envelope stress response modifies peptidoglycan cross-linking via the L,D-transpeptidase LdtD and the novel protein YgaU

Abstract

The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodeling. We sought to characterize the changes that the cell wall undergoes during activation of the Cpx pathway in Escherichia coli. Luminescent reporters of gene expression confirmed that LdtD, a putative l,d-transpeptidase; YgaU, a protein of unknown function; and Slt, a lytic transglycosylase, are upregulated in response to Cpx-inducing conditions. Phosphorylated CpxR binds to the upstream regions of these genes, which contain putative CpxR binding sites, suggesting that regulation is direct. We show that the activation of the Cpx response causes an increase in the abundance of diaminopimelic acid (DAP)-DAP cross-links that involves LdtD and YgaU. Altogether, our data indicate that changes in peptidoglycan structure are part of the Cpx-mediated adaptation to envelope stress and indicate a role for the uncharacterized gene ygaU in regulating cross-linking.

FIG 1

The ldtD, ygaU, and slt promoters are Cpx regulated. All the strains were grown at 30°C. Expression of light from the luminescent reporter genes of ldtD (A and B), ygaU (C and D), and slt (E and F) was measured in the following strains: MC4100, TR10 (cpxA24), TR51 (cpxR::spc), MC4100(pCA-nlpE), and the vector control for NlpE overexpression, MC4100(pCA24N). NlpE overexpression was induced with 0.1 mM IPTG after cultures grown overnight were subcultured (1:40) and grown for 6 h at 30°C. Light was measured at mid- to late log phase (OD₆₀₀ = 0.6 to 0.8) and quantified by dividing the counts per second (CPS) by the OD₆₀₀ of each strain. Each bar represents the mean for three replicates, shown above each bar. Error bars represent the standard deviations. Asterisks indicate that the value is significantly different from that of MC4100 or MC4100(pCA24N) (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). Each experiment was repeated at least three times.

FIG 2

CpxR binds to the promoters of the ldtD, ygaU, and slt genes. (A) Alignments of putative CpxR binding sites in the promoter regions of ldtD, ygaU, and slt. Shown at bottom is the CpxR consensus binding site (http://www.prodoric.de/vfp/index2.php). The numbers indicate the positions of the putative CpxR binding site relative to the transcription start site. (B to F) Various concentrations of purified MBP-CpxR (0 to 200 pmol) were added to 1.5 pmol of the PCR-amplified promoters of degP (positive control) (B), rpoD (negative control) (C), ldtD (D), ygaU (E), and slt (F). Reaction mixtures were loaded onto a 5% nondenaturing TBE polyacrylamide gel (Bio-Rad), electrophoresed in 1× TBE running buffer, and stained with ethidium bromide for visualization.

FIG 3

Absence of ldtD and/or ygaU induces the Cpx pathway. A β-galactosidase assay was carried out on the MC4100 (TR50), MC4100 ΔcpxR (TR70), MC4100 ΔygaU (MBC24), MC4100 ΔldtD (MBC23), and MC4100 ΔldtD ygaU (MBC25) strains carrying a single chromosomal copy of the cpxP-lacZ reporter gene. Cultures grown overnight were subcultured (1:40) and grown until the cells reached late log phase (final OD₆₀₀ = ∼0.7). Each bar represents the mean for three replicates, shown above each bar. Error bars represent the standard deviations. Asterisks represent changes in β-galactosidase levels that are significantly different from those in MC4100 (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). This assay was repeated at least two times, and a representative data set is shown.