Interference of Mycobacterium tuberculosis cell division by Rv2719c, a cell wall hydrolase

Abstract

SummaryThe genetic factors responsible for the regulation of cell division in Mycobacterium tuberculosis are largely unknown. We showed that exposure of M. tuberculosis to DNA damaging agents, or to cephalexin, or growth of M. tuberculosis in macrophages increased cell length and sharply elevated the expression of Rv2719c, a LexA-controlled gene. Overexpression of Rv2719c in the absence of DNA damage or of antibiotic treatment also led to filamentation and reduction in viability both in broth and in macrophages indicating a correlation between Rv2719c levels and cell division. Overproduction of Rv2719c compromised midcell localization of FtsZ rings, but had no effect on the intracellular levels of FtsZ. In vitro, the Rv2719c protein did not interfere with the GTPdependent polymerization activity of FtsZ indicating that the effects of Rv2719c on Z-ring assembly are indirect. Rv2719c protein exhibited mycobacterial murein hydrolase activity that was localized to the N-terminal 110 amino acids. Visualization of nascent peptidoglycan (PG) synthesis zones by probing with fluoresceinated vancomycin (Van-FL) and localization of green fluorescent protein-Rv2719c fusion suggested that the Rv2719c activity is targeted to potential PG synthesis zones. We propose that Rv2719c is a potential regulator of M. tuberculosis cell division and that its levels, and possibly activities, are modulated under a variety of growth conditions including growth in vivo and during DNA damage, so that the assembly of FtsZ-rings, and therefore the cell division, can proceed in a regulated manner.

Polar growth in the Alphaproteobacterial order Rhizobiales

“…Although these modes of cell growth are generally thought to be predominant among rod-shaped bacteria, in Actinobacteria, elongation occurs at the cell poles and requires the polarity determinant protein DivIVA (3)(4)(5)(6)(7)(8). In addition, a striking type of polar growth is found within some Alphaproteobacteria that grow by budding (9) (Fig.…”

Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria

“…Consequently, the surface area that is available for insertion of new cell-wall material increases continuously from birth to next division. In contrast, rod-shaped Actinobacteria, including organisms in the genus Mycobacterium, grow by inserting new cell-wall material exclusively at the cell poles [9][10][11][12][13] . As these cells grow by extending their length while maintaining an approximately constant width, the surface area available for insertion of new cell-wall material at the cell poles does not change appreciably between birth and division.…”

Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes

“…Labeling of wildtype M. smegmatis cells with Bocillin-FL, a fluorescent analog of penicillin, showed distinct foci at midcell and cell poles, which are the potential sites for nascent PG synthesis in mycobacteria (Fig. 6C, panel ii) (6). The Bocillin-FL staining mirrored the GFP-FtsI and GFP-PBPA localization patterns (Fig.…”

“…Therefore, all the cellular localization experiments were carried out in the nonpathogenic M. smegmatis mc 2 155 strain. Cell division proteins in various mycobacterial species have shown a high degree of homology, and M. smegmatis has therefore served as a favorite surrogate host for cell division protein localization studies (6,14,19,20,33). Bocillin-FL, a fluorescent derivative of penicillin V, was used to visualize the overall localization of penicillin-binding proteins in M. smegmatis strains expressing altered CrgA levels.…”

“…Despite this fact, the slow polymerization and weak GTP hydrolysis activities of M. tuberculosis FtsZ combined with the presence of novel proteins such as ChiZ and FipA, which indirectly or directly modulate FtsZ ring assembly, impart uniqueness to the mycobacterial cell division machinery (6,7,45,52). Interaction of FtsZ with phosphorylated FipA is thought to be necessary for productive cell division under oxidative stress (45).…”