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

Abstract

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...

Testing hypotheses for the presence of tRNA genes in mycobacteriophage genomes

“…17,33 Thus, the tRNA genes may only be useful and needed for the expression of some genes and it is the amino acid/ codon usage of these genes that matter. 34 Differences in amino acid and codon usages between the capsid protein and the whole genome of cluster A mycobacteriophages support this argument (Fig. 4 and 5).…”

“…Finally, given the rapid rate of evolution of phage genomes, it is quite possible that tRNA genes have different roles in different clusters. 34 These considerations suggest that much more work needs to be done to understand the presence of tRNA genes in phages.…”

Prophage-mediated defence against viral attack and viral counter-defence

“…Phage lysates typically contained greater than 5x10 9 pfu/ml and were used for plaque assays and DNA extraction. Electron microscopy 42 , genome sequencing, annotation, and analysis were as reported previously 22 . In brief, genomes were annotated using DNAMaster (http://cobamide2.bio.pitt.edu) beginning with an autoannotation with Glimmer and GenMark, followed by manual inspection and revision where necessary for each predicted open reading frame.…”

The Genomes, Proteomes, and Structures of Three Novel Phages That Infect the Bacillus cereus Group and Carry Putative Virulence Factors

“…From genome annotation and nucleotide analysis, Basilisk appeared to be unrelated to JL or Shanette and harbors 138 ORFs and two tRNAs. The presence of tRNAs in all three genomes is common for phages and is expected to compensate for differences in codon usage or the obliteration of host tRNAs (55,56).…”