Genome sequence of Ostreococcus tauri virus OtV-2 throws light on the role of picoeukaryote niche separation in the ocean.

Ostreococcus tauri, a unicellular marine green alga, is the smallest known free-living eukaryote and is ubiquitous in the surface oceans. The ecological success of this organism has been attributed to distinct low- and high-light-adapted ecotypes existing in different niches at a range of depths in the ocean. Viruses have already been characterized that infect the high-light-adapted strains. Ostreococcus tauri virus (OtV) isolate OtV-2 is a large double-stranded DNA algal virus that infects a low-light-adapted strain of O. tauri and was assigned to the algal virus family Phycodnaviridae, genus Prasinovirus. Our working hypothesis for this study was that different viruses infecting high- versus low-light-adapted O. tauri strains would provide clues to propagation strategies that would give them selective advantages within their particular light niche. Sequence analysis of the 184,409-bp linear OtV-2 genome revealed a range of core functional genes exclusive to this low-light genotype and included a variety of unexpected genes, such as those encoding an RNA polymerase sigma factor, at least four DNA methyltransferases, a cytochrome b(5), and a high-affinity phosphate transporter. It is clear that OtV-2 has acquired a range of potentially functional genes from its host, other eukaryotes, and even bacteria over evolutionary time. Such piecemeal accretion of genes is a trademark of large double-stranded DNA viruses that has allowed them to adapt their propagation strategies to keep up with host niche separation in the sunlit layers of the oceanic environment.

Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254.

OBJECTIVES: The aim of this study was to examine the molecular basis for multiple antibiotic and mercury resistance in Canadian isolates of Aeromonas salmonicida subsp. salmonicida. METHODS: Phenotypic and genotypic methods were employed to identify plasmid-associated antibiotic and mercury resistance genes and to determine the organization of those genes in multidrug-resistant (MDR) A. salmonicida isolates. RESULTS: The MDR phenotype was transferable via conjugation using Escherichia coli, Aeromonas hydrophila and Edwardseilla tarda as recipients. Antibiotic and mercury resistance genes were carried by a conjugative IncA/C plasmid. Three distinct antibiotic resistance cassettes were characterized; first a class I integron containing an aadA7 gene encoding for an aminoglycoside-3'-adenyltransferase, the second cassette showed 99.9% nucleotide sequence homology to a cassette previously identified in the Salmonella enterica IncA/C plasmid pSN254, containing floR, tetA, sulII and strA/strB sequences. The third cassette showed 100% nucleotide sequence similarity to a transposon-like element, containing a bla(CMY-2) beta-lactamase in association with sugE and blc sequences. This element is known to be widely distributed among clinical and food-borne Salmonella and other Enterobacteriaceae throughout Asia and the United States. Mercury resistance was linked to the presence of a mer operon that showed 100% nucleotide sequence homology to the mer operon carried by plasmid pSN254. CONCLUSIONS: Each MDR A. salmonicida isolate carried the same plasmid, which was related to plasmid pSN254. This is the first report of plasmid-mediated florfenicol-resistant A. salmonicida in North America. In addition, it is the first report of a plasmid-associated AmpC beta-lactamase sequence in a member of the Aeromonadaceae.

Genomic exploration of individual giant ocean viruses.

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on 'giant viruses' that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

Development of DNA mismatch repair gene, MutS, as a diagnostic marker for detection and phylogenetic analysis of algal Megaviruses.

Megaviruses are generically defined as giant viruses with genomes up to 1.26Mb that infect eukaryotic unicellular protists; they are clearly delineated in DNA polymerase B phylogenetic trees; in addition, common features often include an associated virophage observed during infection; the presence of an amino acyl tRNA synthetase gene; and a nucleic acid mismatch repair protein, MutS gene. The archetypal representative of this evolving putative family is Mimivirus, an opportunistic pathogen of Acanthamoeba spp. originally thought to be a bacterium until its genome sequence was published in 2004. Subsequent analysis of marine metagenomic data revealed Megaviruses are likely ubiquitous on the surface ocean. Analysis of genome sequences of giant viruses isolated from naturally occurring marine protists such as microalgae and a microflagellate grazer, started the expansion of the Megaviridae. Here, we explored the possibility of developing Megavirus specific markers for mutS that could be used in virus molecular ecology studies. MutS is split into 15 different clades representing a wide range of cellular life, and two that contain Megaviruses, clade MutS7 and clade MutS8. We developed specific PCR primers that recognized Megavirus clade MutS8, a clade that we propose discriminates most of the algal Megaviruses. Analysis of seawater off the coast of Maine, US, revealed novel groups of algal Megaviruses that were present in all samples tested. The Megavirus clade MutS8 marker should be considered as a tool to reveal new diversity and distribution of this enigmatic group of viruses.

Phylogenetic affiliations of mesopelagic acantharia and acantharian-like environmental 18S rRNA genes off the southern California coast.

Incomplete knowledge of acantharian life cycles has hampered their study and limited our understanding of their role in the vertical flux of carbon and strontium. Molecular tools can help identify enigmatic life stages and offer insights into aspects of acantharian biology and evolution. We inferred the phylogenetic position of acantharian sequences from shallow water, as well as acantharian-like clone sequences from 500 and 880 m in the San Pedro Channel, California. The analyses included validated acantharian and polycystine sequences from public databases with environmental clone sequences related to acantharia and used Bayesian inference methods. Our analysis demonstrated strong support for two branches of unidentified organisms that are closely related to, but possibly distinct from the Acantharea. We also found evidence of acantharian sequences from mesopelagic environments branching within the chaunacanthid clade, although the morphology of these organisms is presently unknown. HRP-conjugated probes were developed to target Acantharea and phylotypes from Unidentified Clade 1 using Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) on samples collected at 500 m. Our CARD-FISH experiments targeting phylotypes from an unidentified clade offer preliminary glimpses into the morphology of these protists, while a morphology for the aphotic acantharian lineages remains unknown at this time.