Essential cell division enzymes of mycobacteria

Many cases of active tuberculosis are thought to result from the reactivation of dormant Mycobacterium tuberculosis from a prior infection. A family of bacterial extracellular peptidoglycan glycosidases, known as resuscitation-promoting factors (Rpfs), has previously been shown to stimulate growth of dormant mycobacteria, yet the mechanism of reactivation remains unclear. Here we screen a yeast two-hybrid library and report an interaction between RpfB and a putative mycobacterial endopeptidase, which we named Rpf-interacting protein A (RipA). This interaction was confirmed by in vitro and in vivo co-precipitation assays. Both RipA and RpfB localize to the septa of actively growing bacteria by fluorescence microscopy. RipA depletion from M. smegmatis results in a marked decrease in growth and formation of long, branched chains. RipA has hydrolytic activity against several cell wall substrates, which is synergistically enhanced by the addition of RpfB. From a further yeast two-hybrid library we found that RipA interacts with penicillin-binding protein 1 (PBP1), a bifunctional peptidoglycan synthase encoded by ponA1. Depletion of PBP1 from M. smegmatis results in inhibition of growth and formation of abnormally shaped cells, indicating that the ponA1 operon is essential for viability. PBP1 localizes asymmetrically at the poles and occasionally at the septa of dividing cells. Recombinant PBP1 binds to RipA in a co-precipitation assay and inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex. These data reveal a novel potential mechanism of regulating peptidoglycan hydrolysis, ensuring complete septum formation prior to cell division. Furthermore, the synergistic interaction between RipA and RpfB may be important for initiating growth from dormancy.