Septum site placement inMycobacteria- Identification and Characterization of mycobacterial homologues ofEscherichia coliMinD

Abstract

A major virulence trait of Mycobacterium tuberculosis (M. tb) is its ability to enter a dormant state within its human host. Since cell division is intimately linked to metabolic shut down, understanding the mechanism of septum formation and its integration with other events in the division pathway is likely to offer clues to the molecular basis of dormancy. The M. tb genome lacks obvious homologues of several conserved cell division proteins, and this study aimed at identifying and functionally characterising mycobacterial homologues of the E.coli septum site specification protein MinD (Ec MinD). Sequence homology based analyses suggested that the genomes of both M. tb and the saprophyte Mycobacterium smegmatis (M. smegmatis) encode two putative <Ec MinD homologues - Rv1708/MSMEG_3743 and Rv3660c/MSMEG_6171. Both Rv1708 and MSMEG_3743 were observed to fully complement the mini-cell phenotype of the E.coli deltaminDE mutant HL1, but the other homologues only partially complemented the mutant phenotype. Over-expression of MSMEG_3743 but not MSMEG_6171 in M. smegmatis led to cell elongation and a drastic decrease in CFU counts, indicating the essentiality of MSMEG_3743 in cell-division. Sequence analysis of MSMEG_3743 showed a conserved Walker A motif, the functional role of which was confirmed by a radiolabelled ATPase activity assay. Rv1708 was observed to interact with the chromosome associated proteins ScpA and ParB, pointing to a link between its septum formation role and chromosome segregation. Comparative structural analyses showed Rv1708 to be closer in similarity to Ec MinD than Rv3660c. In summary we have demonstrated that Rv1708 and MSMEG_3743 are true mycobacterial homologues of Ec MinD, adding a critical missing piece to the mycobacterial cell division puzzle.