Genome and Ecology of a Novel Alteromonas Podovirus, ZP6, Representing a New Viral Genus, Mareflavirus.

Alteromonas is a ubiquitous, abundant, copiotrophic and phytoplankton-associated marine member of the Gammaproteobacteria with a range extending from tropical waters to polar regions and including hadal zones. Here, we describe a novel Alteromonas phage, ZP6, that was isolated from surface coastal waters of Qingdao, China. ZP6 contains a linear, double-stranded, 38,080-bp DNA molecule with 50.1% G+C content and 47 putative open reading frames (ORFs). Three auxiliary metabolic genes were identified, encoding metal-dependent phosphohydrolase, diaminopurine synthetase, and nucleotide pyrophosphohydrolase. The first two ORFs facilitate the replacement of adenine (A) by diaminopurine (Z) in phage genomes and help phages to evade attack from host restriction enzymes. The nucleotide pyrophosphohydrolase enables the host cells to stop programmed cell death and improves the survival rate of the host in a nutrient-depleted environment. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analysis revealed that ZP6 is most closely related to Enhodamvirus but with low similarity (shared genes, <30%, and average nucleotide sequence identity, <65%); it is distinct from other bacteriophages. Together, these results suggest that ZP6 could represent a novel viral genus, here named Mareflavirus. Combining its ability to infect Alteromonas, its harboring of a diaminopurine genome-biosynthetic system, and its representativeness of an understudied viral group, ZP6 could be an important and novel model system for marine virus research. IMPORTANCEAlteromonas is an important symbiotic bacterium of phytoplankton, but research on its bacteriophages is still at an elementary level. Our isolation and genome characterization of a novel Alteromonas podovirus, ZP6, identified a new viral genus of podovirus, namely, Mareflavirus. The ZP6 genome, with a diaminopurine genome-biosynthetic system, is different from those of other isolated Alteromonas phages and will bring new impetus to the development of virus classification and provide important insights into novel viral sequences from metagenomic data sets.

A Novel Broad Host Range Phage Infecting Alteromonas.

Bacteriophages substantially contribute to bacterial mortality in the ocean and play critical roles in global biogeochemical processes. Alteromonas is a ubiquitous bacterial genus in global tropical and temperate waters, which can cross-protect marine cyanobacteria and thus has important ecological benefits. However, little is known about the biological and ecological features of Alteromonas phages (alterophages). Here, we describe a novel alterophage vB_AmeP-R8W (R8W), which belongs to the Autographiviridae family and infects the deep-clade Alteromonas mediterranea. R8W has an equidistant and icosahedral head (65 ± 1 nm in diameter) and a short tail (12 ± 2 nm in length). The genome size of R8W is 48,825 bp, with a G + C content of 40.55%. R8W possesses three putative auxiliary metabolic genes encoding proteins involved in nucleotide metabolism and DNA binding: thymidylate synthase, nucleoside triphosphate pyrophosphohydrolase, and PhoB. R8W has a rapid lytic cycle with a burst size of 88 plaque-forming units/cell. Notably, R8W has a wide host range, such that it can infect 35 Alteromonas strains; it exhibits a strong specificity for strains isolated from deep waters. R8W has two specific receptor binding proteins and a compatible holin-endolysin system, which contribute to its wide host range. The isolation of R8W will contribute to the understanding of alterophage evolution, as well as the phage-host interactions and ecological importance of alterophages.

A Novel Phage Infecting Alteromonas Represents a Distinct Group of Siphophages Infecting Diverse Aquatic Copiotrophs.

Bacteriophages play critical roles in impacting microbial community succession both ecologically and evolutionarily. Although the majority of phage genetic diversity has been increasingly unveiled, phages infecting members of the ecologically important genus Alteromonas remain poorly understood. Here, we present a comprehensive analysis of a newly isolated alterophage, vB_AcoS-R7M (R7M), to characterize its life cycle traits, genomic features, and putative evolutionary origin. R7M harbors abundant genes identified as host-like auxiliary metabolic genes facilitating viral propagation. Genomic analysis suggested that R7M is distinct from currently known alterophages. Interestingly, R7M was found to share a set of similar characteristics with a number of siphophages infecting diverse aquatic opportunistic copiotrophs. We therefore proposed the creation of one new subfamily (Queuovirinae) to group with these evolutionarily related phages. Notably, tail genes were less likely to be shared among them, and baseplate-related genes varied the most. In-depth analyses indicated that R7M has replaced its distal tail with a Rhodobacter capsulatus gene transfer agent (RcGTA)-like baseplate and further acquired a putative receptor interaction site targeting Alteromonas. These findings suggest that horizontal exchanges of viral tail adsorption apparatuses are widespread and vital for phages to hunt new hosts and to adapt to new niches. IMPORTANCE The evolution and ecology of phages infecting members of Alteromonas, a marine opportunistic genus that is widely distributed and of great ecological significance, remain poorly understood. The present study integrates physiological and genomic evidence to characterize the properties and putative phage-host interactions of a newly isolated Alteromonas phage, vB_AcoS-R7M (R7M). A taxonomic study reveals close evolutionary relationships among R7M and a number of siphophages infecting various aquatic copiotrophs. Their similar head morphology and overall genetic framework suggest their putative common ancestry and the grouping of a new viral subfamily. However, their major difference lies in the viral tail adsorption apparatuses and the horizontal exchanges of which possibly account for variations in host specificity. These findings outline an evolutionary scenario for the emergence of diverse viral lineages of a shared genetic pool and give insights into the genetics and ecology of viral host jumps.

Characterization and Genome Analysis of a Novel Marine Alteromonas Phage P24.

Although Alteromonas is ubiquitous in the marine environment, very little is known about Alteromonas phages, with only ten, thus far, being isolated and reported on. In this study, a novel double-stranded DNA phage, Alteromonas phage P24, which infects Alteromonas macleodii, was isolated from the coastal waters off Qingdao. Alteromonas phage P24 has a siphoviral morphology, with an icosahedral head, 61 ± 1 nm in diameter, and a tail length of 105 ± 1 nm. Alteromonas phage P24 contains lipids. It has an optimal temperature and pH for growth of 20℃ and 5-7, respectively. A one-step growth curve shows a latent period of 55 min, a rise period of 65 min, and an average burst size of approximately 147 virions per cell. Alteromonas phage P24 has the genome of 46,945 bp with 43.80% GC content and 74 open reading frames (ORFs) without tRNA. The results of the phylogenetic tree, based on the mcp and terL genes, show that Alteromonas phage P24 is closely related to Aeromonas phage phiARM81ld. Meanwhile, phylogenetic analysis based on the whole genome of P24 indicates that it forms a unique viral sub-cluster within Siphoviridae. This study contributes to the understanding of the genomic characteristics and the virus-host interactions of Alteromonas phages.

Characterization and Genome Analysis of a Novel Alteromonas Phage JH01 Isolated from the Qingdao Coast of China.

A novel Alteromonas phage JH01, with the host strain identified to be Alteromonas marina SW-47(T), was isolated from the Qingdao coast during the summer of 2017. Transmission electron microscopy analysis showed that phage JH01 can be categorized into the Siphoviridae family, with an icosahedral head of 62 ± 5 nm and a long contractile tail of 254 ± 10 nm. The bioinformatic analysis shows that this phage consists of a linear, double-stranded 46,500 bp DNA molecule with a GC content of 44.39%, and 58 ORFs with no tRNA genes. The ORFs are classified into four groups, including phage packaging, phage structure, DNA replication and regulation, and hypothetical protein. The phylogenetic tree, constructed using neighbor-joining analysis, shows that phage JH01 has altitudinal homology with some Vibrio and Pseudoalteromonas phage B8b. Comparative analysis reveals the high similarity between phage JH01 and phage B8b. Additionally, our study of phage JH01 provides useful information for further research on the interaction between Alteromonas phages and their hosts.

Characterization and Genome Sequence of Marine Alteromonas gracilis Phage PB15 Isolated from the Yellow Sea, China.

A novel marine Alteromonas gracilis siphovirus, phage PB15, was isolated from the surface water of the Yellow Sea in August 2015. It has a head diameter of 58 ± 5 nm head and a contractile tail approximately 105 ± 10 nm in length, and overall, the morphology suggests that PB15 belongs to the family Siphoviridae. PB15 phage is stable at over the temperature range 0-60 °C. The best MOI of these phage was 0.1, and infectivity decreased above 60 °C. The results suggest that phage is stable at pH value ranging between 3.0 and 11.0. Chloroform test shows that PB15 is not a lipid-containing phage. A one-step growth curve with a strain of A. gracilis gave a latent period of 16 min and rise period of 24 min and burst size of 60 PFU/cell. Genomic analysis of PB15 reveals a genome size of 37,333 bp with 45.52% G+C content, and 61 ORFs. ORF sequences accounted for 30.36% of the genome sequence. There is no obvious similarity between PB15 and other known phages by genomic comparison using the BLASTN tool in the NCBI database.

Transcription of bacteriophage PM2.

Transcription of bacteriophage PM2 after infection of Alteromonas espejiana BAL-31 was examined. A wave of PM2 late transcription was observed 22 to 44 min after infection. This transcription was chloramphenicol- and rifampicin-sensitive. Regions of PM2 DNA transcribed with high efficiency were determined by DNA--RNA hybridization and Southern's technique.