The mycobacteriophage Ms6 encodes a chaperone‐like protein involved in the endolysin delivery to the peptidoglycan

Catalão, Maria João; Gil, Filipa; Moniz-Pereira, José; Pimentel, Madalena

doi:10.1111/j.1365-2958.2010.07239.x

Cited by 55 publications

(104 citation statements)

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“…We propose that the second of these smaller genes (Bongo gp37, PegLeg gp37, and Rey gp42) is the holin, with each having two predicted transmembrane domains, although they are not related at the sequence level to any other mycobacteriophage proteins. The first small protein (e.g., Bongo gp36, 78 amino acids) contains an N-terminal predicted transmembrane domain, and its role is unclear, although it could perform a chaperone-like role in lysis, as proposed for gp1 of phage Ms6 (38). The lysin A proteins have domain structures corresponding to organization A (Org-A) (39), although the only other similar mycobacteriophage lysin is that encoded by the singleton phage Patience (gp41; 36% amino acid identity).…”

Section: Resultsmentioning

confidence: 99%

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotypes

Pope

Anders

Baird

et al. 2014

J Virol

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Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc 2 155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCEThe bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.T he bacteriophage population is large, dynamic, old, and genetically diverse (1). Over 1,000 phage genomes have been sequenced, and the majority are double-stranded DNA (dsDNA) tailed phages, classified morphologically in the order Caudovirales. The genomes of dsDNA tailed phages vary in length from ϳ20 kbp to over 500 kbp (2) and typically contain 20 to 30 genes encoding virion structure and assembly functions, genes coding for DNA and nucleotide metabolism, a lysis cassette, and regulatory systems. The genomes of temperate phages typically encode repressors, contain operators, and frequently include systems for phage genome integration. However, with the exception of the few well-studied phage prototypes, the majority of genes carried by phages are of unknown function (1). Mycobacteriophages-vir...

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Section: Resultsmentioning

confidence: 99%

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotypes

Pope

Anders

Baird

et al. 2014

J Virol

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“…1A) (12). Recently, we have reported that export of the Ms6 endolysin (LysA) is holin independent and that LysA translocation across the cytoplasmic membrane is assisted by Gp1, a chaperone-like protein, encoded by the first gene of the Ms6 lysis cassette (2). These data, together with the absence of a lysis phenotype when Gp4 was coexpressed in E. coli with Ms6 endolysin (4), even when both proteins were shown to be expressed at detectable levels (M. J. Catalão, unpublished data), led us to reanalyze the amino acid sequence of Ms6 Gp4.…”

Section: Resultsmentioning

confidence: 99%

“…Construction of Ms6 mutants was performed using bacteriophage recombineering of electroporated DNA (BRED) in M. smegmatis. Recombineering substrates and BRED strategy were done as described previously (2,19). Briefly, for deletion of the Ms6 gp4, gp5, or gp4 and gp5 genes (yielding Ms6 ⌬gp4 , Ms6 ⌬gp5 , or Ms6 ⌬gp4 gp5 , respectively), 100-bp oligonucleotides, Pr⌬gp4, Pr⌬gp5, or Pr⌬gp4gp5, that have 50 bp of homology upstream and downstream of the region to be deleted, were extended by PCR using two 75-bp extender primers, PrExt⌬gp4fwd/PrExt⌬gp4rv, PrExt⌬gp5fwd/ PrExt⌬gp5rv, or PrExt⌬gp4fwd/PrExt⌬gp5rv, respectively, which have 25 bp of homology to the ends of the 100-mer and add an additional 50 bp of homology on either end.…”

Section: Methodsmentioning

confidence: 99%

“…In addition to the endolysin (lysA) and a holin-like gene (gp4), three accessory lysis genes restricted to mycobacteriophages were also identified: gp1, lysB, and gp5. The gp1 gene was recently identified as encoding a chaperone-like protein that specifically interacts with the N-terminal region of LysA and is involved in its delivery to the peptidoglycan in a holin-independent manner (2). The Ms6 holin-like protein, encoded by gp4 (previously named hol), shares some structural characteristics with class II holins, which are usually hydrophobic in nature and small, with a hydrophilic carboxy-terminal domain and two potential transmembrane domains (TMD1 and TMD2).…”

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confidence: 99%

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Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

et al. 2011

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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.

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“…Holins allow the endolysins to gain access to the cell wall, where they exert their actions by cleaving various bonds in the peptidoglycan polymer, depending on the type of endolysin (4,13). Genes encoding holin proteins and their target peptidoglycan hydrolases have been identified in a wide variety of Gram-negative and Gram-positive bacteria and their phages (1,(14)(15)(16)(17). While possible homologues are present in archaea (see holin families 1.E.3, 1.E.14, and 1.E.43 in TCDB), the functions of these proteins are not yet defined.…”

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confidence: 99%

Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

2015

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bHolins form pores in the cytoplasmic membranes of bacteria for the primary purpose of releasing endolysins that hydrolyze the cell wall and induce cell death. Holins are encoded within bacteriophage genomes, where they promote cell lysis for virion release, and within bacterial genomes, where they serve a diversity of potential or established functions. These include (i) release of gene transfer agents, (ii) facilitation of programs of differentiation such as those that allow sporulation and spore germination, (iii) contribution to biofilm formation, (iv) promotion of responses to stress conditions, and (v) release of toxins and other proteins. There are currently 58 recognized families of holins and putative holins with members exhibiting between 1 and 4 transmembrane ␣-helical spanners, but many more families have yet to be discovered. Programmed cell death in animals involves holin-like proteins such as Bax and Bak that may have evolved from bacterial holins. Holin homologues have also been identified in archaea, suggesting that these proteins are ubiquitous throughout the three domains of life. Phage-mediated cell lysis of dualmembrane Gram-negative bacteria also depends on outer membrane-disrupting "spanins" that function independently of, but in conjunction with, holins and endolysins. In this minireview, we provide an overview of their modes of action and the first comprehensive summary of the many currently recognized and postulated functions and uses of these cell lysis systems. It is anticipated that future studies will result in the elucidation of many more such functions and the development of additional applications.

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The mycobacteriophage Ms6 encodes a chaperone‐like protein involved in the endolysin delivery to the peptidoglycan

Cited by 55 publications

References 42 publications

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotypes

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotypes

Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

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