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.
