Novel Cluster AZ Arthrobacter phages Powerpuff, Lego, and YesChef exhibit close functional relationships with Microbacterium phages.

Bacteriophages exhibit a vast spectrum of relatedness and there is increasing evidence of close genomic relationships independent of host genus. The variability in phage similarity at the nucleotide, amino acid, and gene content levels confounds attempts at quantifying phage relatedness, especially as more novel phages are isolated. This study describes three highly similar novel Arthrobacter globiformis phages-Powerpuff, Lego, and YesChef-which were assigned to Cluster AZ using a nucleotide-based clustering parameter. Phages in Cluster AZ, Microbacterium Cluster EH, and the former Microbacterium singleton Zeta1847 exhibited low nucleotide similarity. However, their gene content similarity was in excess of the recently adopted Microbacterium clustering parameter, which ultimately resulted in the reassignment of Zeta1847 to Cluster EH. This finding further highlights the importance of using multiple metrics to capture phage relatedness. Additionally, Clusters AZ and EH phages encode a shared integrase indicative of a lysogenic life cycle. In the first experimental verification of a Cluster AZ phage's life cycle, we show that phage Powerpuff is a true temperate phage. It forms stable lysogens that exhibit immunity to superinfection by related phages, despite lacking identifiable repressors typically required for lysogenic maintenance and superinfection immunity. The ability of phage Powerpuff to undergo and maintain lysogeny suggests that other closely related phages may be temperate as well. Our findings provide additional evidence of significant shared phage genomic content spanning multiple actinobacterial host genera and demonstrate the continued need for verification and characterization of life cycles in newly isolated phages.

BlueFeather, the singleton that wasn't: Shared gene content analysis supports expansion of Arthrobacter phage Cluster FE.

Bacteriophages (phages) exhibit high genetic diversity, and the mosaic nature of the shared genetic pool makes quantifying phage relatedness a shifting target. Early parameters for clustering of related Mycobacteria and Arthrobacter phage genomes relied on nucleotide identity thresholds but, more recently, clustering of Gordonia and Microbacterium phages has been performed according to shared gene content. Singleton phages lack the nucleotide identity and/or shared gene content required for clustering newly sequenced genomes with known phages. Whole genome metrics of novel Arthrobacter phage BlueFeather, originally designated a putative singleton, showed low nucleotide identity but high amino acid and gene content similarity with Arthrobacter phages originally assigned to Clusters FE and FI. Gene content similarity revealed that BlueFeather shared genes with these phages in excess of the parameter for clustering Gordonia and Microbacterium phages. Single gene analyses revealed evidence of horizontal gene transfer between BlueFeather and phages in unique clusters that infect a variety of bacterial hosts. Our findings highlight the advantage of using shared gene content to study seemingly genetically isolated phages and have resulted in the reclustering of BlueFeather, a putative singleton, as well as former Cluster FI phages, into a newly expanded Cluster FE.

Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages.

The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45-68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.

Molecular Analysis of Arthrobacter Myovirus vB_ArtM-ArV1: We Blame It on the Tail.

This is the first report on a myophage that infects Arthrobacter A novel virus, vB_ArtM-ArV1 (ArV1), was isolated from soil using Arthrobacter sp. strain 68b for phage propagation. Transmission electron microscopy showed its resemblance to members of the family Myoviridae: ArV1 has an isometric head (∼74 nm in diameter) and a contractile, nonflexible tail (∼192 nm). Phylogenetic and comparative sequence analyses, however, revealed that ArV1 has more genes in common with phages from the family Siphoviridae than it does with any myovirus characterized to date. The genome of ArV1 is a linear, circularly permuted, double-stranded DNA molecule (71,200 bp) with a GC content of 61.6%. The genome includes 101 open reading frames (ORFs) yet contains no tRNA genes. More than 50% of ArV1 genes encode unique proteins that either have no reliable identity to database entries or have homologues only in Arthrobacter phages, both sipho- and myoviruses. Using bioinformatics approaches, 13 ArV1 structural genes were identified, including those coding for head, tail, tail fiber, and baseplate proteins. A further 6 ArV1 ORFs were annotated as encoding putative structural proteins based on the results of proteomic analysis. Phylogenetic analysis based on the alignment of four conserved virion proteins revealed that Arthrobacter myophages form a discrete clade that seems to occupy a position somewhat intermediate between myo- and siphoviruses. Thus, the data presented here will help to advance our understanding of genetic diversity and evolution of phages that constitute the order CaudoviralesIMPORTANCE Bacteriophages, which likely originated in the early Precambrian Era, represent the most numerous population on the planet. Approximately 95% of known phages are tailed viruses that comprise three families: Podoviridae (with short tails), Siphoviridae (with long noncontractile tails), and Myoviridae (with contractile tails). Based on the current hypothesis, myophages, which may have evolved from siphophages, are thought to have first emerged among Gram-negative bacteria, whereas they emerged only later among Gram-positive bacteria. The results of the molecular characterization of myophage vB_ArtM-ArV1 presented here conform to the aforementioned hypothesis, since, at a glance, bacteriophage vB_ArtM-ArV1 appears to be a siphovirus that possesses a seemingly functional contractile tail. Our work demonstrates that such "chimeric" myophages are of cosmopolitan nature and are likely characteristic of the ecologically important soil bacterial genus Arthrobacter.

An optimized enrichment technique for the isolation of Arthrobacter bacteriophage species from soil sample isolates.

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobacter sp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

Isolation and characterization of vB_ArS-ArV2 - first Arthrobacter sp. infecting bacteriophage with completely sequenced genome.

This is the first report on a complete genome sequence and biological characterization of the phage that infects Arthrobacter. A novel virus vB_ArS-ArV2 (ArV2) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Based on transmission electron microscopy, ArV2 belongs to the family Siphoviridae and has an isometric head (∼63 nm in diameter) with a non-contractile flexible tail (∼194×10 nm) and six short tail fibers. ArV2 possesses a linear, double-stranded DNA genome (37,372 bp) with a G+C content of 62.73%. The genome contains 68 ORFs yet encodes no tRNA genes. A total of 28 ArV2 ORFs have no known functions and lack any reliable database matches. Proteomic analysis led to the experimental identification of 14 virion proteins, including 9 that were predicted by bioinformatics approaches. Comparative phylogenetic analysis, based on the amino acid sequence alignment of conserved proteins, set ArV2 apart from other siphoviruses. The data presented here will help to advance our understanding of Arthrobacter phage population and will extend our knowledge about the interaction between this particular host and its phages.

Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined. Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B. lactofermentum, and a sequence almost identical to attB was found to be present in these three strains. In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome. This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510.