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 was 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. Of these, Rv1708/MSMEG_3743 were found to be the true homologues, through complementation of the E. coli ∆minDE mutant HL1, overexpression studies, and structural comparisons. Rv1708 and MSMEG_3743 fully complemented the mini-cell phenotype of HL1, and over-expression of MSMEG_3743 in M. smegmatis led to cell elongation and a drastic decrease in c.f.u. counts, indicating its essentiality in cell-division. MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif. Interaction of Rv1708 with the chromosome associated proteins ScpA and ParB, implied 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 identify Rv1708 and MSMEG_3743 to be homologues of Ec MinD, adding a critical missing piece to the mycobacterial cell division puzzle.
Subject(s)
Escherichia coli Proteins , Mycobacterium tuberculosis , Humans , Escherichia coli/genetics , Escherichia coli/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Cell Division/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolismABSTRACT
The major virulence trait of Mycobacterium tuberculosis is its ability to enter a latent state in the face of robust host immunity. Clues to the molecular basis of latency can emerge from understanding the mechanism of cell division, beginning with identification of proteins involved in this process. Using complementation of Escherichia coli mutants, we functionally annotated M. tuberculosis and Mycobacterium smegmatis homologs of divisome proteins FtsW and AmiC. Our results demonstrate that E. coli can be used as a surrogate model to discover mycobacterial cell division genes, and should prove invaluable in delineating the mechanisms of this fundamental process in mycobacteria.