Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ⁷⁰-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA

Abstract

A mycobacteriophage Ms6 strong promoter region (P lys ) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem 70 -like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region P lys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that ␤-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation. Double-stranded DNA (dsDNA) bacteriophages synthesize a mureinolytic enzyme, known as an endolysin, during late gene expression of the replication cycle, enabling the release of phage progeny. Phage-encoded lysins have several kinds of mureinolytic activities directed against the covalent linkages that maintain the cell wall integrity: (i) glycosylase and transglycosylase activity, targeting the glycosidic linkages; (ii) Nacetylmuramoyl-L-alanine amidase activity; and (iii) endopeptidase activity targeting the oligopeptide cross-links (17, 31).To degrade the cell wall of the host, a second factor, designated holin, is required. The holin is a hydrophobic membrane protein that forms pores or lesions in the cell membrane through which the murein hydrolase is released to the periplasm and gains access to the peptidoglycan substrate. Such a dual-component lysis system has recently been discovered in many bacteriophages of both gram-negative and grampositive bacteria (15,17,22,25,27,32). The synthesis of the bacteriophage holin (protein S) is tightly controlled at the transcriptional, translational, and posttranslational levels because it determines the time of lysis. In phage , the lysis genes are the first genes of the late operon, which also encodes the structural proteins of the phage particle. Transcription of the cell lysis...

The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity

“…The gene lysB, which is localized downstream of the endolysin gene lysA, and immediately upstream of the holin gene hol, is 996 bp in length, and encodes a 332 aa protein (Garcia et al, 2002). The presence of the pentapeptide Gly-Tyr-Ser-Gln-Gly within the amino acid sequence matches the consensus motif Gly-X-Ser-X-Gly characteristic of lipolytic enzymes, and led us to search for lipolytic activity.…”

“…Growth and viability of the recombinant strain E. coli JM109 (pMP302) was not affected, even after the addition of 2 % CHCl 3 (data not shown). It has been shown for other phages that expression of a bacteriophage lysin gene in E. coli causes cellular lysis after permeabilization of the plasma membrane with chloroform (Chandry et al, 1997;Henrich et al, 1995); this effect is also observed with the lysin LysA of mycobacteriophage Ms6 (Garcia et al, 2002). The result indicates that LysB is not an additional endolysin.…”

“…The lysis module of the mycobacteriophage Ms6, a temperate phage that infects Mycobacterium smegmatis (Portugal et al, 1989), has previously been identified, and consists of five genes (Garcia et al, 2002). In addition to the endolysin (lysA) and the holin (hol) genes, the Ms6 lytic cassette comprises three additional genes whose functions…”

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).…”

Insights into the regulation of bacteriophage endolysin: multiple means to the same end

“…Indeed, in order to identify the promoter for the endolysin gene of mycobacteriophage Ms6, the desired region was fused to the lacZ reporter. It was then demonstrated that lacZ was expressed from two s 70 -like promoters, providing evidence that transcription of endolysin genes can be carried out by host transcription factors (Garcia et al, 2002). It would be of immense interest to explore the involvement of various transcription factors in the expression of endolysin genes.…”