Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

Abstract

The mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) bacteriophage which, in addition to the predicted endolysin (LysA)-holin (Gp4) lysis system, encodes three additional proteins within its lysis module: Gp1, LysB, and Gp5. Ms6 Gp4 was previously described as a class II holin-like protein. By analysis of the amino acid sequence of Gp4, an N-terminal signal-arrest-release (SAR) domain was identified, followed by a typical transmembrane domain (TMD), features which have previously been observed for pinholins. A second putative holin gene (gp5) encoding a protein with a predicted single TMD at the N-terminal region was identified at the end of the Ms6 lytic operon. Neither the putative class II holin nor the single TMD polypeptide could trigger lysis in pairwise combinations with the endolysin LysA in Escherichia coli. One-step growth curves and single-burst-size experiments of different Ms6 derivatives with deletions in different regions of the lysis operon demonstrated that the gene products of gp4 and gp5, although nonessential for phage viability, appear to play a role in controlling the timing of lysis: an Ms6 mutant with a deletion of gp4 (Ms6 ⌬gp4 ) caused slightly accelerated lysis, whereas an Ms6 ⌬gp5 deletion mutant delayed lysis, which is consistent with holin function. Additionally, cross-linking experiments showed that Ms6 Gp4 and Gp5 oligomerize and that both proteins interact. Our results suggest that in Ms6 infection, the correct and programmed timing of lysis is achieved by the combined action of Gp4 and Gp5.

Molecular Dissection of Phage Endolysin

“…Earlier studies with lysins have utilized chloroform to make the plasma membrane permeable that then allows the diffusion of lysins to the cell wall peptidoglycan and its digestion (53)(54)(55). Several other studies have followed the lysin effect after its co-expression with Holin (25,56). Our methodology developed here will not only help in studying the activity of these proteins in detail but will also aid in identifying new proteins from other bacteriophages when this method of expression is followed in a suitable host.…”

“…However, whether it has similar function in D29 or other mycobacteriophages remains to be elucidated. For example, in Ms6, holin is not essential for the translocation of endolysin from cytoplasm to periplasm (25). The D29 and the Ms6 holin were found to have lethal effects on Escherichia coli cells upon expression (25).…”

“…For example, in Ms6, holin is not essential for the translocation of endolysin from cytoplasm to periplasm (25). The D29 and the Ms6 holin were found to have lethal effects on Escherichia coli cells upon expression (25). Very recently, the first transmembrane helix of D29 holin was synthesized and studied by carrying out detailed biophysical analysis (26).…”

The Endolysin-Binding Domain Encompasses the N-Terminal Region of the Mycobacteriophage Ms6 Gp1 Chaperone

“…Gp1 specifically binds the N-terminal 60 amino acids of the Ms6 endolysin and allows the enzyme access to its substrate, the peptidoglycan, by somehow facilitating its secretion across the cytoplasmic membrane independently of Ms6 holin-like protein activity (1,2). Even though lysA was shown to encode two proteins (3), the 384-amino-acid lysin (lysin 384 ) and lysin 241 , with lysin 241 resulting from the use of an internal, in-frame translation initiation site within the lysA gene, only lysin 384 interacts with Gp1 (1).…”

“…Besides determination of the Gp1 regions that interact with LysA, investigation of its contribution to the lysis phenotype is also important. Wild-type Gp1 and mutant proteins were coexpressed with wild-type lysin 384 in Escherichia coli, under the control of a T5 promoter by using derivative plasmids of pMP320 (a pQE30 [Qiagen] derivative that harbors gp1 and lysA genes) (2). Deletions of the N-terminal, central, and C-terminal Gp1 regions were made as described above, generating plasmids pMP320⌬1-84bpgp1 , pMP320⌬85-157bpgp1, and pMP320⌬158-221bpgp1 (Table 1).…”