The effects of mycobacterial RmlA perturbation on cellular dNTP pool, cell morphology, and replication stress in Mycobacterium smegmatis.

Rita Hirmondó,Dániel Molnár,Ármin Horváth,György Török,Liem Nguyen,Judit Tóth,Olivier Neyrolles

doi:10.1371/journal.pone.0263975

Abstract

The concerted action of DNA replication and cell division has been extensively investigated in eukaryotes. Well demarcated checkpoints have been identified in the cell cycle, which provides the correct DNA stoichiometry and appropriate growth in the progeny. In bacteria, which grow faster and less concerted than eukaryotes, the linkages between cell elongation and DNA synthesis are unclear. dTTP, one of the canonical nucleotide-building blocks of DNA, is also used for cell wall biosynthesis in mycobacteria. We hypothesize that the interconnection between DNA and cell wall biosynthesis through dTTP may require synchronization of these processes by regulating dTTP availability. We investigated growth, morphology, cellular dNTP pool, and possible signs of stress in Mycobacterium smegmatis upon perturbation of rhamnose biosynthesis by the overexpression of RmlA. RmlA is a cell wall synthetic enzyme that uses dTTP as the precursor for cross-linking the peptidoglycan with the arabinogalactan layers by a phosphodiester bond in the mycobacterial cell wall. We found that RmlA overexpression results in changes in cell morphology, causing cell elongation and disruption of the cylindrical cell shape. We also found that the cellular dTTP pool is reduced by half in RmlA overexpressing cells and that this reduced dTTP availability does not restrict cell growth. We observed 2-6-fold increases in the gene expression of replication and cell wall biosynthesis stress factors upon RmlA overexpression. Using super-resolution microscopy, we found that RmlA, acting to crosslink the nascent layers of the cell wall, localizes throughout the whole cell length in a helical pattern in addition to the cellular pole.

Highlights

The unique intricate cell wall impermeable for most antibiotics is a specific hallmark of mycobacteria
Successful cloning was verified with the sequencing of the appropriate region of the plasmid. 0.5–0.5 μg of the appropriate plasmids were electroporated into electrocompetent wild type (WT) [26] or PknG(-) [18] M. smegmatis strain according to Table 1
To decipher how dTTP metabolism and cell wall biosynthesis affect each other, we investigated the cellular function of RmlA, the cell wall biosynthetic enzyme that potentially links these two processes together using dTTP in the first step of the rhamnose biosynthetic pathway

Summary

Introduction

The unique intricate cell wall impermeable for most antibiotics is a specific hallmark of mycobacteria. The galactan region of the arabinogalactan layer is bound to the peptidoglycan layer via a phosphodiester linkage of the α-L-rhamnopyranose-(1!3)-α-D-GlcNAc-1-phosphate disaccharide [5] This linker, composed of a rhamnosyl residue, a sugar not found in humans, is critical to the structural integrity of the mycobacterial cell wall (Fig 1), as well as for the viability and pathogenicity of pathogenic mycobacteria [6, 7]. Consistent with this, the RmlA enzyme (D-glucose-1-phosphate thymidylyltransferase), which catalyzes the first step of rhamnose biosynthesis, is essential for bacterial growth [12]. The importance of this enzyme for mycobacterial viability, together with the absence of the rhamnose pathway in humans, make this enzyme a potentially valid drug target for the development of RmlA-related anti-mycobacterial drugs for tuberculosis treatment [13]

Methods

Results

Conclusion