Diversification of LytM Protein Functions in Polar Elongation and Cell Division of Agrobacterium tumefaciens.

Wanda M Figueroa-Cuilan,Caroline M Dunn,Andrew Yowell,Pamela J B Brown,Amelia M Randich,Gustavo Santiago-Collazo

doi:10.3389/fmicb.2021.729307

Wanda M Figueroa-Cuilan, Caroline M Dunn + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2021.729307

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Abstract

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

Highlights

In bacteria, the peptidoglycan (PG) cell wall plays an essential role in maintaining cell shape, protecting bacteria from environmental stressors, and preventing cell lysis (Scheffers and Pinho, 2005; Vollmer et al, 2008; Cava et al, 2013; Cameron et al, 2015; Ruiz, 2016)
The inactivation of the catalytic site has been well-documented among various studies of degenerate” LytM (dLytM) factors in the Proteobacteria and it has been shown in various members that these factors activate cognate amidases rather than directly hydrolyzing peptide bonds themselves (Uehara et al, 2009; Stohl et al, 2016; Yang et al, 2018; Gurnani Serrano et al, 2021)
We determined that A. tumefaciens putatively encodes two enzymatically active LytM factors and two inactive factors

Summary

Introduction

The peptidoglycan (PG) cell wall plays an essential role in maintaining cell shape, protecting bacteria from environmental stressors, and preventing cell lysis (Scheffers and Pinho, 2005; Vollmer et al, 2008; Cava et al, 2013; Cameron et al, 2015; Ruiz, 2016). While the bacterial PG cell wall is necessary and sufficient to determine bacterial cell shape, expansion, and separation of the cell wall requires enzymatic action (Uehara and Bernhardt, 2011). The final steps of cell division require the coordinated activity of cell wall amidases, endopeptidases, carboxypeptidases, lytic transglycosylases, and regulators of these hydrolyzes called LytM factors to enable cell separation (Heidrich et al, 2001; Peters et al, 2011; Vermassen et al, 2019; Do et al, 2020). In E. coli and V. cholerae, endopeptidases that have lost enzymatic activity are termed “degenerate” LytM (dLytM) factors and function as regulators of amidase activity

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