Characterization of the Role of the Rpf Motif in Mycobacteriophage Tape Measure Proteins

In order to inject their DNA into the bacterial cytoplasm and establish infection, bacteriophages must ensure their genetic material successfully traverses both the bacterial membrane(s) and the layer of peptidoglycan surrounding the host cell. Phages accomplish this in a variety of ways, and some have virion-associated murein hydrolase enzymes that facilitate this process, particularly in conditions where the peptidoglycan is highly cross-linked. Phages that infect the mycobacteria must also contend with these barriers to infection, as well an impermeable layer of mycolic acids that decorates the cell surface; however, the mechanisms by which they do this are mostly unknown. In this regard, three small sequence motifs have been identified within mycobacteriophage tape measure proteins (TMPs) − extended molecules that span the tail lumen and determine its length − at least two of which have similarity to host proteins with muralytic activity. This suggests that phages may utilize regions of the TMP, which because of its location within the tail might be uniquely primed for host interaction, to facilitate localized peptidoglycan hydrolysis and DNA injection. The focus of this study is the motif found in the TMPs of mycobacteriophages Barnyard and Giles that has identity to a group of bacterial proteins known as resuscitation promoting factors (Rpfs). These Rpf proteins stimulate growth of non-growing bacteria and seem to exert their activity by cleaving inert peptidoglycan in the cell wall. Notably, the Barnyard Rpf Motif is contained within a 70 kDa C-terminal cleavage product of TMP, which appears to be cell wall- and/or membrane-associated during infection. Further, mycobacteria expressing TMP fragments containing this motif show aberrant behavior in culture and on solid media, and hybrid proteins in which the Rpf domain of the Micrococcus luteus Rpf protein is replaced with either of the phage motifs have muralytic activity in vivo. A recombineering-based method for generating mutations on lytically replicating mycobacteriophages has been developed and utilized to make multiple mutations in the Giles TMP motifs. Mutant phages infect host cells in late-stationary phase with a reduced efficiency, an observation that further supports a role for these motifs in cell wall hydrolysis during infection