Mycobacteriophage D29 holin C‐terminal region functionally assists in holin aggregation and bacterial cell death

Abstract

Holins are phage-encoded small transmembrane proteins that perforate the bacterial cytoplasmic membrane. In most cases, this process allows the phage-encoded peptidoglycan hydrolases to act on the cell wall, resulting in host cell lysis and phage release. We report a detailed functional characterization of Mycobacterium phage D29 gp11 coding for a putative holin that, upon expression, rapidly kills both Escherichia coli and Mycobacterium smegmatis. We dissected Gp11 by making several deletions and expressing them in E. coli. The shortening of Gp11 from its C-terminus results in diminished cytotoxicity and smaller holes. Evidently, the two transmembrane domains (TMDs) present at the N-terminus of Gp11 are incapable of integrating into the cytoplasmic membrane and do not show toxicity. Interestingly, the fusion of two TMDs and a small C-terminal region that bears the coiled-coil motif resulted in restoration of the cell killing ability of the protein. We further show that the second TMD is dispensable in protein toxicity because its deletion does not abolish Gp11-mediated cell death. We conclude that Gp11 C-terminal region is necessary but not sufficient for toxicity. These results shed light on a yet undiscovered role of Gp11 C-terminal region that will help clarify the mechanism of holin-mediated membrane perforation. Finally, we abolish the toxicity of Gp11 using a specific Gly to Asp substitution in the putative loop region of the protein; the mutant protein may help to clarify how holin functions in mycobacteriophage D29.

Robust High Throughput Real-Time Monitoring Assay for the Specific Screening of Bacterial Cell Envelope Inhibitors

“…Similar increase in fluorescence is not observed when an inactive holin mutant, HolG28D, is expressed (Fig. 2); we have previously shown that HolG28D is incapable of causing bacterial cell death (Kamilla and Jain, 2016). These studies thus confirm that the sytox green assay can be used to examine the properties of membrane pore forming proteins.…”

“…The expression of D29 mycobacteriophage holin and its mutant was achieved as described previously (Kamilla and Jain, 2016). Briefly, E. coli BL21(DE3) cells carrying expression plasmids were grown at 37°C in LB-broth supplemented with 100 µg ml -1 ampicillin.…”

“…Upon its production, holin is known to form holes in the cell membrane of bacteria (O'Donnell et al, 1992;Kamilla and Jain, 2016). Holins along with the peptidoglycan hydrolases are required by the phages to lyse the cells to release their progeny from the host.…”

Solvation driven conformational transitions in the second transmembrane domain of mycobacteriophage holin

“…It has recently been proposed that the C-terminal cytosolic domain of holin forms a coiledcoil motif that promotes holin-mediated bacterial cytotoxicity through oligomerization. 51 Hence, it is likely that the second transmembrane domain facilitates the functioning of the other domains of holin: (i) conformational switch of the first transmembrane domain, and (ii) oligomerization of the C-terminal domain. Through this study, we find that the second transmembrane domain of Mycobacteriophage D29 holin can adopt multiple conformations (a-helical and PPII-like forms) in solution.…”

“…TM2 does not possess the conserved GX 2/3 G motif required for helix dimerization, 54,55 nor does it show any propensity for a coiled-coil motif. 51 Hence, it is unclear whether TM2 can form homo-or hetero-oligomers in vivo. We do find that TM2 has a short hydrophobic segment (Supporting Information Figure S8), which might facilitate TM2 localization in the membrane.…”