A Novel Subcluster of Closely Related Bacillus Phages with Distinct Tail Fiber/Lysin Gene Combinations.

Bacteriophages (phages) are the most numerous entities on Earth, but we have only scratched the surface of describing phage diversity. We isolated seven Bacillus subtilis phages from desert soil in the southwest United States and then sequenced and characterized their genomes. Comparative analyses revealed high nucleotide and amino acid similarity between these seven phages, which constitute a novel subcluster. Interestingly, the tail fiber and lysin genes of these phages seem to come from different origins and carry out slightly different functions. These genes were likely acquired by this subcluster of phages via horizontal gene transfer. In conjunction with host range assays, our data suggest that these phages are adapting to hosts with different cell walls.

Genomic and phenotypic signatures of bacteriophage coevolution with the phytopathogen Pseudomonas syringae.

The rate and trajectory of evolution in an obligate parasite is critically dependent on those of its host(s). Adaptation to a genetically homogeneous host population should theoretically result in specialization, while adaptation to an evolving host population (i.e., coevolution) can result in various outcomes including diversification, range expansion, and/or local adaptation. For viruses of bacteria (bacteriophages, or phages), our understanding of how evolutionary history of the bacterial host(s) impacts viral genotypic and phenotypic evolution is currently limited. In this study, we used whole genome sequencing and two different metrics of phage impacts to compare the genotypes and phenotypes of lytic phages that had either coevolved with or were repeatedly passaged on an unchanging (ancestral) strain of the phytopathogen Pseudomonas syringae. Genomes of coevolved phages had more mutations than those of phages passaged on a constant host, and most mutations were in genes encoding phage tail-associated proteins. Phages from both passaging treatments shared some phenotypic outcomes, including range expansion and divergence across replicate populations, but coevolved phages were more efficient at reducing population growth (particularly of sympatric coevolved hosts). Genotypic similarity correlated with infectivity profile similarity in coevolved phages, but not in phages passaged on the ancestral host. Overall, while adaptation to either host type (coevolving or ancestral) led to divergence in phage tail proteins and infectivity patterns, coevolution led to more rapid molecular changes that increased bacterial killing efficiency and had more predictable effects on infectivity range. Together, these results underscore the important role of hosts in driving viral evolution and in shaping the genotype-phenotype relationship.

Forty Years without Family: Three Novel Bacteriophages with High Similarity to SPP1 Reveal Decades of Evolutionary Stasis since the Isolation of Their Famous Relative.

SPP1, an extensively studied bacteriophage of the Gram-positive Bacillus subtilis, is a model system for the study of phage-host interactions. Despite progress in the isolation and characterization of Bacillus phages, no previously fully sequenced phages have shared more than passing genetic similarity to SPP1. Here, we describe three virulent phages very similar to SPP1; SPP1 has greater than 80% nucleotide sequence identity and shares more that 85% of its protein coding genes with these phages. This is remarkable, given more than 40 years between the isolation of SPP1 and these phages. All three phages have somewhat larger genomes and more genes than SPP1. We identified a new putative gene in SPP1 based on a conserved sequence found in all phages. Gene conservation connotes purifying selection and is observed in structural genes and genes involved in DNA metabolism, but also in genes of unknown function, suggesting an important role in phage survival independent of the environment. Patterns of divergence point to genes or gene domains likely involved in adaptation to diverse hosts or different environments. Ultimately, comparative genomics of related phages provides insight into the long-term selective pressures that affect phage-bacteria interactions and alter phage genome content.

Complete Genome Sequences of Two Temperate Bacillus subtilis Phages Isolated at Tumamoc Hill Desert Laboratory.

Bacteriophages are important in structuring bacterial communities, including desert soils dominated by Bacillus species. Here, we describe two genetically similar temperate phages isolated on a Bacillus subtilis strain from soil in Tucson, Arizona. Their double-stranded DNA (dsDNA) genomes contain 98 and 102 genes, with a set of 4 genes being found in only one phage.

Comparative Genomics of Six Lytic Bacillus subtilis Phages from the Southwest United States.

Background: Despite their importance to microbial dynamics involving Bacillus subtilis, we have a limited understanding of the diversity of phages that can lyse this model organism. Materials and Methods: Phages were isolated from soil samples collected from various sites in the southwest U.S. deserts on a wild B. subtilis strain. Their genomes were assembled, characterized, and bioinformatically compared. Results: Six Siphoviruses with high nucleotide and amino acid similarity to each other (>80%) but very limited similarity to phages currently in GenBank were isolated. These phages have double-stranded DNA genomes (55,312 to 56,127 bp) with 86-91 putative protein coding genes, and a low GC content. Comparative genomics reveal differences in loci encoding proteins that are putatively involved in bacterial adsorption with evidence for genomic mosaicism and a possible role for small genes. Conclusions: A comparative approach provides insights into phage evolution, including the role of indels in protein folding.

Complete Genome Sequences of Four Phages of the Horse Chestnut Phyllosphere.

Bacteriophages play important roles in determining bacterial communities, including plant microbiota. Here, we describe four lytic phages, three Siphoviridae and one Podoviridae, isolated from four different bacterial species found on the leaves of horse chestnut trees. Their double-stranded DNA (dsDNA) genomes range from 39,095 to 46,062 bp and contain 51 to 70 genes.

Genome Sequences of Erwinia Phyllophages AH04 and AH06.

Although crucial in shaping bacterial communities, few bacteriophages of the phyllosphere have been described. We provide genome data for two Myoviridae phages, AH04 and AH06, isolated on Erwinia billingiae strains. AH04 shares limited genetic similarity with previously described phages, while AH06 shares over 75% similarity with various Erwinia phages.

Complete Genome Sequence of the Pantoea Phage AH07.

Bacteriophages of the phyllosphere have not been extensively described, despite their role in bacterial communities on this plant organ. Here, we describe a temperate Pantoea phage, AH07, that was isolated from the leaves of horse chestnut trees. The 37,859-bp linear double-stranded DNA genome contains 58 putative genes, including an integration cassette.

Testing hypotheses for the presence of tRNA genes in mycobacteriophage genomes.

The presence of tRNA genes in bacteriophages has been explained on the basis of codon usage (tRNA genes are retained in the phage genome if they correspond to codons more common in the phage than in its host) or amino acid usage (independent of codon, the amino acid corresponding to the retained tRNA gene is more common in the phage genome than in the bacterial host). The existence of a large database of sequenced mycobacteriophages, isolated on the common host Mycobacterium smegmatis, allows us to test the above hypotheses as well as explore other hypotheses for the presence of tRNA genes. Our analyses suggest that amino acid rather than codon usage better explains the presence of tRNA genes in mycobacteriophages. However, closely related phages that differ in the presence of tRNA genes in their genomes are capable of lysing the common bacterial host and do not differ in codon or amino acid usage. This suggests that the benefits of having tRNA genes may be associated with either growth in the host or the ability to infect more hosts (i.e., host range) rather than simply infecting a particular host.

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.

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.