Cell Separation in Vibrio cholerae Is Mediated by a Single Amidase Whose Action Is Modulated by Two Nonredundant Activators

Abstract

Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB's localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks.

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

“…E. coli contains three amidases, AmiA, AmiB, and AmiC, which are regulated by NlpD (AmiC) and EnvC (AmiA and AmiB) (27). The situation is slightly more streamlined in V. cholerae, which has a single amidase, AmiB, which is regulated by NlpD and EnvC (8). Because of the redundancy of these regulatory genes in E. coli and V. cholerae, deletion of nlpD has only minor, if any, effects on cellular morphology (8,27,28).…”

“…The situation is slightly more streamlined in V. cholerae, which has a single amidase, AmiB, which is regulated by NlpD and EnvC (8). Because of the redundancy of these regulatory genes in E. coli and V. cholerae, deletion of nlpD has only minor, if any, effects on cellular morphology (8,27,28). A recent report on LytM proteins in H. influenzae showed that an nlpD mutant not only exhibited defects in cell separation and growth rate but also showed increased outer membrane vesicle release, indicating a role for NlpD in membrane stability (9).…”

“…In E. coli, these LytM-like proteins have been shown to regulate the amidases AmiA to AmiC, to contain a degenerate LytM domain possessing only two of four LytM active sites, and to have no PG hydrolytic activity themselves (5). The rod-shaped bacterium Vibrio cholerae also contains a single amidase, AmiB, which is controlled by NlpD and EnvC (8). Haemophilus influenzae contains three LytM-like factors, YebA, NlpD, and Env (9).…”

Evolutionary convergence in experimental Pseudomonas populations

“…Genome-sequencing data available from that study (of line 6B 4 ) suggested candidate genes and Sanger sequencing was used to find the mutational causes. In 23 out of 24 strains a mutation was found in PFLU1301 (nlpD), which encodes a lipoprotein that in E. coli (Uehara et al, 2010), Vibrio cholerae (Moll et al, 2014) and Yersinia pestis (Tidhar et al, 2009) is an activator of AmiC (N-acetylmuramoyl-L-alanine amidase) hydrolase activity required for proper cell division: loss-offunction mutations in nlpD in these species exhibit a similar cellular morphology as observed for the CC types and can be complemented by expression in trans. In all 23 cases the mutation in nlpD was identical-a single Q189* (CAG-4TAG) nonsense mutation.…”

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

“…The Gram-negative bacterium V. cholerae has curved rod morphology, and HPLC analyses of strain N16961 previously detected shorter glycan strands than in E. coli (26,55,56). We performed a similar survey of morphology and peptidoglycan content from strains CA401, MO10, MZ02, MAK757, and 0139, which cover the El Tor (MO10, MAK757) and Classical (CA401) biogroups, and the O139 (MO10), O1 (CA401, MAK757), and non-O1, non-O139 (MZO2) serotypes.…”