title: 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.
authors.affiliation: Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Bhopal, India.
authors.forename: Soumya
authors.initials: S
authors.lastname: Kamilla
authors.affiliation: Microbiology and Molecular Biology Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Bhopal, India.
authors.forename: Vikas
authors.initials: V
authors.lastname: Jain
mesh_terms: D004926:Escherichia coli / Q000166:cytology* / Q000821:virology; D050296:Microbial Viability; D009160:Mycobacteriophages / Q000737:chemistry* / Q000472:pathogenicity; D020102:Mycobacterium smegmatis / Q000166:cytology* / Q000821:virology*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014764:Viral Proteins
keywords: bacterial cell death; coiled-coil motif; endolysin; membrane perforation; transmembrane domain