Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes.

UNLABELLED: 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. IMPORTANCE: The 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.

Isolation, characterization, and comparative genomic analysis of a phage infecting high-level aminoglycoside-resistant (HLAR) Enterococcus faecalis.

Enterococcus is a genus of Gram-positive bacteria that are commensal to the gastrointestinal tracts of humans but some species have been increasingly implicated as agents of nosocomial infections. The increase in infections and the spread of antibiotic-resistant strains have contributed to renewed interest in the discovery of Enterococcus phages. The aims of this study were (1) the isolation, characterization, and genome sequencing of a phage capable of infecting an antibiotic-resistant E. faecalis strain, and (2) the comparative genomic analysis of publicly-available Enterococcus phages. For this purpose, multiple phages were isolated from wastewater treatment plant (WWTP) influent using a high-level aminoglycoside-resistant (HLAR) E. faecalis strain as the host. One phage, phiNASRA1, demonstrated a high lytic efficiency (∼97.52%). Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) showed that phiNASRA1 belongs to the Siphoviridae family of double-stranded DNA viruses. The phage was approximately 250 nm in length and its complete genome (40,139 bp, 34.7% GC) contained 62 open reading frames (ORFs). Phylogenetic comparisons of phiNASRA1 and 31 publicly-available Enterococcus phages, based on the large subunit terminase and portal proteins, grouped phage by provenance, size, and GC content. In particular, both phylogenies grouped phages larger than 100 kbp into distinct clades. A phylogeny based on a pangenome analysis of the same 32 phages also grouped phages by provenance, size, and GC content although agreement between the two single-locus phylogenies was higher. Per the pangenome phylogeny, phiNASRA1 was most closely related to phage LY0322 that was similar in size, GC content, and number of ORFs (40,139 and 40,934 bp, 34.77 and 34.80%, and 60 and 64 ORFs, respectively). The pangenome analysis did illustrate the high degree of sequence diversity and genome plasticity as no coding sequence was homologous across all 32 phages, and even 'conserved' structural proteins (e.g., the large subunit terminase and portal proteins) were homologous in no more than half of the 32 phage genomes. These findings contribute to a growing body of literature devoted to understanding phage biology and diversity. We propose that this high degree of diversity limited the value of the single-locus and pangenome phylogenies. By contrast, the high degree of homology between phages larger than 100 kbp suggests that pangenome analyses of more similar phages is a viable method for assessing subclade diversity. Future work is focused on validating phiNASRA1 as a potential therapeutic agent to eradicate antibiotic-resistant E. faecalis infections in an animal model.

Comparative genomics of Cluster O mycobacteriophages.

Mycobacteriophages--viruses of mycobacterial hosts--are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages--Corndog, Catdawg, Dylan, Firecracker, and YungJamal--designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8-9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.