Abstract
The integrity of the cell envelope of E. coli relies on the concerted activity of multi‐protein machineries that synthesize the peptidoglycan (PG) and the outer membrane (OM). Our previous work found that the depletion of lipopolysaccharide (LPS) export to the OM induces an essential PG remodeling process involving LD‐transpeptidases (LDTs), the glycosyltransferase function of PBP1B and the carboxypeptidase PBP6a. Consequently, cells with defective OM biogenesis lyse if they lack any of these PG enzymes. Here we report that the morphological defects, and lysis associated with a ldtF mutant with impaired LPS transport, are alleviated by the loss of the predicted OM‐anchored lipoprotein ActS (formerly YgeR). We show that ActS is an inactive member of LytM‐type peptidoglycan endopeptidases due to a degenerated catalytic domain. ActS is capable of activating all three main periplasmic peptidoglycan amidases, AmiA, AmiB, and AmiC, which were previously reported to be activated only by EnvC and/or NlpD. Our data also suggest that in vivo ActS preferentially activates AmiC and that its function is linked to cell envelope stress.
Highlights
Gram-negative bacteria have a thin peptidoglycan (PG) layer that is surrounded by an asymmetric outer membrane (OM) which protects cells from many toxic molecules and antibiotics (Huang et al, 2008; Silhavy et al, 2010)
In E. coli the majority of peptide cross-links in PG is of 4-3 (DD) type and formed by penicillin-binding proteins (PBPs) (Egan et al, 2020; Typas et al, 2012), but a minority of 3-3 (LD) cross-links are formed by LD-transpeptidases (LDTs) (Magnet et al, 2008)
LdtD plays a major role in survival under these conditions, but the glycosyltransferase of PBP1B, its activator LpoB, and the carboxypeptidase PBP6a are needed, and it was proposed that these enzymes repair together defects in the PG that arise upon OM assembly stress (Morè et al, 2019)
Summary
Okuda et al, 2016; Sperandeo et al, 2019). The Lpt machinery works as a single device and the depletion of any of its component halts LPS export, resulting in a growth arrest and the formation of short chains of unseparated cells (Ruiz et al, 2008; Sperandeo et al, 2008). ActS interacts with AmiC (Figure 5a,b) and activates AmiC better than NlpD (Figure 4b,c), and the LytM domain of ActS has the highest sequence similarity to that of NlpD (Figure 1), suggesting that ActS might have a preference for AmiC in the cell To test this hypothesis, the pGS100-actS plasmid was introduced into the ΔenvC ΔamiC and ΔnlpD ΔamiAB mutants, and the chaining phenotype was assessed. We assessed whether the lysis phenotype of the araBplptC ΔactS mutant can be rescued by the deletion of amiC and found that lack of AmiC does not protect from lysis under nonpermissive conditions (Figure S6a) This result is consistent with data reported in the accompanying manuscript (Mueller et al, 2021) where AmiA is shown to be sufficient for normal cell separation under neutral and acidic conditions. We show that deletion of nlpD does not suppress the lysis phenotype of lptC-depleted actS-deleted cells (Figure S6b), further supporting the notion that there is a specific functional connection between LdtF and ActS
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