Yet More Evidence of Collusion: a New Viral Defense System Encoded by Gordonia Phage CarolAnn.

Temperate phages play important roles in the physiology of their bacterial hosts and establish a lysogenic relationship with the host through which prophage-expressed genes confer new phenotypes. A key phenotype is prophage-mediated defense against heterotypic viral attack, in which temperate phages collude with their bacterial host to prevent other phages from attacking, sometimes with exquisite specificity. Such defense systems have been described in Pseudomonas and Mycobacterium phages but are likely widespread throughout the microbial community. Here, we describe a novel prophage-mediated defense system encoded by Gordonia phage CarolAnn, which defends against infection by unrelated phages grouped in cluster CZ. CarolAnn genes 43 and 44 are coexpressed with the repressor and are necessary and sufficient to confer defense against phage Kita and its close relatives. Kita and these relatives are targeted through Kita gene 53, a gene that is of unknown function but which is the location of defense escape mutations that overcome CarolAnn defense. Expression of Kita gene 53 is toxic to Gordonia terrae in the presence of CarolAnn genes 43 and 44, suggesting that defense may be mediated by an abortive infection type of mechanism. CarolAnn genes 43 and 44 are distant relatives of mycobacteriophage Sbash genes 31 and 30, respectively, which also confer viral defense but use a different targeting system.IMPORTANCE Prophage-mediated viral defense systems play a key role in microbial dynamics, as lysogeny is established relatively efficiently, and prophage-expressed genes can strongly inhibit lytic infection of other, unrelated phages. Demonstrating such defense systems in Gordonia terrae suggests that these systems are widespread and that there are a multitude of different systems with different specificities for the attacking phages.

More Evidence of Collusion: a New Prophage-Mediated Viral Defense System Encoded by Mycobacteriophage Sbash.

The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 1023 phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships.

The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable.IMPORTANCE Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of Gordonia shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host Mycobacterium smegmatis, although one group of Gordonia phages is more closely related to mycobacteriophages than to the other Gordonia phages. Phage genome sequence space remains largely unexplored, but further isolation and genomic comparison of phages targeted at related groups of hosts promise to reveal pathways of bacteriophage evolution.

Complete Genome Sequences of 38 Gordonia sp. Bacteriophages.

We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity.

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

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defence promotes phage co-evolution.

Genome Sequence of Gordonia Phage BetterKatz.

BetterKatz is a bacteriophage isolated from a soil sample collected in Pittsburgh, Pennsylvania using the host Gordonia terrae 3612. BetterKatz's genome is 50,636 bp long and contains 75 predicted protein-coding genes, 35 of which have been assigned putative functions. BetterKatz is not closely related to other sequenced Gordonia phages.

Genome Sequences of Gordonia Phages Bowser and Schwabeltier.

Gordonia phages Bowser and Schwabeltier are newly isolated phages infecting Gordonia terrae 3612. Bowser and Schwabeltier have similar siphoviral morphologies and their genomes are related to each other, but not to other phages. Their lysis cassettes are atypically situated among virion tail genes, and Bowser encodes two tyrosine integrases.

Genome Sequence of Gordonia Phage Emalyn.

Emalyn is a newly isolated bacteriophage of Gordonia terrae 3612 and has a double-stranded DNA genome 43,982 bp long with 67 predicted protein-encoding genes, 32 of which we can assign putative functions. Emalyn has a prolate capsid and has extensive nucleotide similarity with several previously sequenced phages.

Genome Sequences of Gordonia Phages Hotorobo, Woes, and Monty.

Hotorobo, Woes, and Monty are newly isolated bacteriophages of Gordonia terrae 3612. The three phages are related, and their genomes are similarly sized (76,972 bp, 73,752 bp, and 75,680 bp for Hotorobo, Woes, and Monty, respectively) and organized. They have extremely long tails and among the longest tape measure protein genes described to date.

Genome Sequences of Gordonia Phages BaxterFox, Kita, Nymphadora, and Yeezy.

Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae, isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%.

Genome Sequences of Gordonia terrae Bacteriophages Phinally and Vivi2.

Bacteriophages Phinally and Vivi2 were isolated from soil from Pittsburgh, Pennsylvania, USA, using host Gordonia terrae 3612. The Phinally and Vivi2 genomes are 59,265 bp and 59,337 bp, respectively, and share sequence similarity with each other and with GTE6. Fewer than 25% of the 87 to 89 putative genes have predictable functions.

Genome Sequence of Gordonia Phage Yvonnetastic.

Gordonia bacteriophage Yvonnetastic was isolated from soil in Pittsburgh, PA, using Gordonia terrae 3612 as a host. Yvonnetastic has siphoviral morphology and a genome of 98,136 bp, with 198 predicted protein-coding genes and five tRNA genes. Yvonnetastic does not share substantial sequence similarity with other sequenced bacteriophage genomes.

Genome Sequence of Gordonia Bacteriophage Lucky10.

Lucky10 is a newly isolated phage of Gordonia terrae 3612 that was recovered from a soil sample in Pittsburgh, PA. Lucky10 has siphoviral morphology and a double-stranded DNA (dsDNA) genome of 42,979 bp, with 70 predicted protein-coding genes. Lucky10 shows little similarity to previously reported Gordonia phages.

Genome Sequences of Gordonia terrae Phages Attis and SoilAssassin.

Attis and SoilAssassin are two closely related bacteriophages isolated on Gordonia terrae 3612 from separate soil samples in Pittsburgh, PA. The Attis and SoilAssassin genomes are 47,881 bp and 47,880 bp, respectively, and have 74 predicted protein-coding genes, including toxin-antitoxin systems, but no tRNAs.

Genome Sequences of Gordonia Bacteriophages Obliviate, UmaThurman, and Guacamole.

We describe three newly isolated phages-Obliviate, UmaThurman, and Guacamole-that infect Gordonia terrae 3612. The three genomes are related to one another but are not closely related to other previously sequenced phages or prophages. The three phages are predicted to use integration-dependent immunity systems as described in several mycobacteriophages.

Genome Sequences of Gordonia terrae Phages Benczkowski14 and Katyusha.

Bacteriophages Katyusha and Benczkowski14 are newly isolated phages that infect Gordonia terrae 3612. Both have siphoviral morphologies with isometric heads and long tails (500 nm). The genomes are 75,380 bp long and closely related, and the tape measure genes (9 kbp) are among the largest to be identified.

Genome Sequences of Cluster G Mycobacteriophages Cambiare, FlagStaff, and MOOREtheMARYer.

Mycobacteriophages Cambiare, FlagStaff, and MOOREtheMARYer are newly isolated phages of Mycobacterium smegmatis mc(2) 155 recovered from soil samples in Pittsburgh, PA. All three genomes are closely related to cluster G mycobacteriophages but differ sufficiently in nucleotide sequence and gene content to warrant division of cluster G into several subclusters.

Genome Sequences of Mycobacteriophages AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo, New Members of Cluster B.

AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo are newly isolated phages of Mycobacterium smegmatis mc(2)155 discovered in Pittsburgh, Pennsylvania, USA. All five phages share nucleotide similarity with cluster B mycobacteriophages but span considerable diversity with Corofin and OrangeOswald in subcluster B3, AlanGrant and Vincenzo in subcluster B4, and Baee in subcluster B5.

Genome Sequence of Mycobacteriophage Phayonce.

Mycobacteriophage Phayonce is a newly isolated phage recovered from a soil sample in Pittsburgh, PA, using Mycobacterium smegmatis mc(2)155 as a host. Phayonce's genome is 49,203 bp long and contains 77 protein-coding genes, 23 of them having predicted functions. Phayonce shares a strong similarity in nucleotide sequence with phages of cluster P.