by Rita Hirmondó, Ármin Horváth, Dániel Molnár, György Török, Liem Nguyen, Judit Tóth
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.