Strong seasonality and interannual recurrence in marine myovirus communities.

The temporal community dynamics and persistence of different viral types in the marine environment are still mostly obscure. Polymorphism of the major capsid protein gene, g23, was used to investigate the community composition dynamics of T4-like myoviruses in a North Atlantic fjord for a period of 2 years. A total of 160 unique operational taxonomic units (OTUs) were identified by terminal restriction fragment length polymorphism (TRFLP) of the gene g23. Three major community profiles were identified (winter-spring, summer, and autumn), which resulted in a clear seasonal succession pattern. These seasonal transitions were recurrent over the 2 years and significantly correlated with progression of seawater temperature, Synechococcus abundance, and turbidity. The appearance of the autumn viral communities was concomitant with the occurrence of prominent Synechococcus blooms. As a whole, we found a highly dynamic T4-like viral community with strong seasonality and recurrence patterns. These communities were unexpectedly dominated by a group of persistently abundant viruses.

Genomic sequence and analysis of EhV-99B1, a new coccolithovirus from the Norwegian fjords.

Coccolithoviruses are giant dsDNA viruses that infect Emiliania huxleyi, the most ubiquitous marine microalga. Here, we present the genome of the latest coccolithovirus strain to be sequenced, EhV-99B1, and compare it with two other coccolithovirus genomes (EhV-86 and EhV-163). EhV-99B1 shares a pairwise nucleotide identity of 98% with EhV-163 (the two strains were isolated from the same Norwegian fjord but in different years), and just 96.5% with EhV-86 (isolated in the same spring as EhV-99B1 but in the English Channel). We confirmed and extended the list of relevant genomic differences between these EhVs from the Norwegian fjord and EhVs from the English Channel, namely the removal/insertions of: a phosphate permease, an endonuclease, a transposase, and two specific tRNAs. As a whole, this study provided new clues and insights into the diversity and mechanisms driving the evolution of these large oceanic viruses, in particular those processes involving selfish genetic elements.

Counterintuitive carbon-to-nutrient coupling in an Arctic pelagic ecosystem.

Predicting the ocean's role in the global carbon cycle requires an understanding of the stoichiometric coupling between carbon and growth-limiting elements in biogeochemical processes. A recent addition to such knowledge is that the carbon/nitrogen ratio of inorganic consumption and release of dissolved organic matter may increase in a high-CO(2) world. This will, however, yield a negative feedback on atmospheric CO(2) only if the extra organic material escapes mineralization within the photic zone. Here we show, in the context of an Arctic pelagic ecosystem, how the fate and effects of added degradable organic carbon depend critically on the state of the microbial food web. When bacterial growth rate was limited by mineral nutrients, extra organic carbon accumulated in the system. When bacteria were limited by organic carbon, however, addition of labile dissolved organic carbon reduced phytoplankton biomass and activity and also the rate at which total organic carbon accumulated, explained as the result of stimulated bacterial competition for mineral nutrients. This counterintuitive 'more organic carbon gives less organic carbon' effect was particularly pronounced in diatom-dominated systems where the carbon/mineral nutrient ratio in phytoplankton production was high. Our results highlight how descriptions of present and future states of the oceanic carbon cycle require detailed understanding of the stoichiometric coupling between carbon and growth-limiting mineral nutrients in both autotrophic and heterotrophic processes.

Phylogenetic analysis of members of the Phycodnaviridae virus family, using amplified fragments of the major capsid protein gene.

Algal viruses are considered ecologically important by affecting host population dynamics and nutrient flow in aquatic food webs. Members of the family Phycodnaviridae are also interesting due to their extraordinary genome size. Few algal viruses in the Phycodnaviridae family have been sequenced, and those that have been have few genes in common and low gene homology. It has hence been difficult to design general PCR primers that allow further studies of their ecology and diversity. In this study, we screened the nine type I core genes of the nucleocytoplasmic large DNA viruses for sequences suitable for designing a general set of primers. Sequence comparison between members of the Phycodnaviridae family, including three partly sequenced viruses infecting the prymnesiophyte Pyramimonas orientalis and the haptophytes Phaeocystis pouchetii and Chrysochromulina ericina (Pyramimonas orientalis virus 01B [PoV-01B], Phaeocystis pouchetii virus 01 [PpV-01], and Chrysochromulina ericina virus 01B [CeV-01B], respectively), revealed eight conserved regions in the major capsid protein (MCP). Two of these regions also showed conservation at the nucleotide level, and this allowed us to design degenerate PCR primers. The primers produced 347- to 518-bp amplicons when applied to lysates from algal viruses kept in culture and from natural viral communities. The aim of this work was to use the MCP as a proxy to infer phylogenetic relationships and genetic diversity among members of the Phycodnaviridae family and to determine the occurrence and diversity of this gene in natural viral communities. The results support the current legitimate genera in the Phycodnaviridae based on alga host species. However, while placing the mimivirus in close proximity to the type species, PBCV-1, of Phycodnaviridae along with the three new viruses assigned to the family (PoV-01B, PpV-01, and CeV-01B), the results also indicate that the coccolithoviruses and phaeoviruses are more diverged from this group. Phylogenetic analysis of amplicons from virus assemblages from Norwegian coastal waters as well as from isolated algal viruses revealed a cluster of viruses infecting members of the prymnesiophyte and prasinophyte alga divisions. Other distinct clusters were also identified, containing amplicons from this study as well as sequences retrieved from the Sargasso Sea metagenome. This shows that closely related sequences of this family are present at geographically distant locations within the marine environment.

Discovery of a dsRNA virus infecting the marine photosynthetic protist Micromonas pusilla.

We report the isolation of the first double-stranded (ds) RNA virus in the family Reoviridae that infects a protist (microalga Micromonas pusilla, Prasinophyceae). The dsRNA genome was composed of 11 segments ranging between 0.8 and 5.8 kb, with a total size of approximately 25.5 kb. The virus (MpRNAV-01B) could not be assigned to the genus level because host type, genome size, and number of segments smaller than 2 kb did not correspond to either of the two existing 11-segmented dsRNA genera Rotavirus and Aquareovirus. MpRNAV-01B has a particle size of 65-80 nm, a narrow host range, a latent period of 36 h, and contains five major proteins (120, 95, 67, 53, and 32 kDa). MpRNAV-01B was stable to freeze-thawing, resistant to chloroform, ether, nonionic detergents, chelating and reducing agents. The virus was inactivated at temperatures above 35 degrees C and by ionic detergent, ethanol, acetone, and acidic conditions (pH 2-5).

Isolation and characterization of two viruses with large genome size infecting Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae).

Two lytic viruses specific for Chrysochromulina ericina (Prymnesiophyceae) and for Pyramimonas orientalis (Prasinophyceae) were isolated from Norwegian coastal waters in June 1998. The lytic cycle was 14-19 h for both viruses; the burst size was estimated at 1800-4100 viruses per host cell for the Chrysochromulina virus and 800-1000 for the Pyramimonas virus. Thin sections of infected cells show that both viruses replicate in the cytoplasm and that they have a hexagonal cross section, indicating icosahedral symmetry. The Chrysochromulina virus had a particle size of 160 nm and a genome size of 510 kbp; the size of the major polypeptide was 73 kDa. The Pyramimonas virus had a particle size of 220 x 180 nm and a genome size of 560 kbp; the size of the major polypeptide was 44 kDa. The genome sizes of these viruses are among the largest ever reported for viruses and they are larger than the minimum required for cellular life. The Chrysochromulina virus clone CeV-01B and the Pyramimonas virus clone PoV-01B described in this study have several properties in common with other viruses infecting microalgae, suggesting that they belong to the Phycodnaviridae.

Abundance and diversity of Archaea in heavy-metal-contaminated soils.

The impact of heavy-metal contamination on archaean communities was studied in soils amended with sewage sludge contaminated with heavy metals to varying extents. Fluorescent in situ hybridization showed a decrease in the percentage of Archaea from 1.3% +/- 0.3% of 4', 6-diamidino-2-phenylindole-stained cells in untreated soil to below the detection limit in soils amended with heavy metals. A comparison of the archaean communities of the different plots by denaturing gradient gel electrophoresis revealed differences in the structure of the archaean communities in soils with increasing heavy-metal contamination. Analysis of cloned 16S ribosomal DNA showed close similarities to a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species.

Novel techniques for analysing microbial diversity in natural and perturbed environments.

Molecular techniques were applied for analysing the entire bacterial community, including both the cultivated and non-cultivated part of the community. DNA was extracted from samples of soils and sediments, and a combination of different molecular methods were used to investigate community structure and diversity in these environments. Reassociation of sheared and thermally denatured DNA in solution was used to measure the total genetical diversity. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of rRNA genes gave information about changes in the numerically dominating bacterial populations. Hybridisation with phylogenetic group specific probes, and sequencing provided information about the affiliation of the bacterial populations. Using DNA reassociation analysis we demonstrated that bacterial communities in pristine soil and sediments may contain more than 10,000 different bacterial types. The diversity of the total soil community was at least 200 times higher than the diversity of bacterial isolates from the same soil. This indicates that the culturing conditions select for a distinct subpopulation of the bacteria present in the environment. Molecular methods were applied to monitor the effects of perturbations due to antropogenic activities and pollution on microbial communities. Our investigations show that agricultural management, fish farming and pollution may lead to profound changes in the community structure and a reduction in the bacterial diversity.

Analysis of broad-scale differences in microbial community composition of two pristine forest soils.

Broad-scale differences in soil microbial community composition were analyzed in two contrasting soils using DNA reassociation and % G + C profiles for analysis on the community-level, and filter- and whole cell hybridization techniques for a coarse-level characterization of larger phylogenetic groups of bacteria. Reassociation analysis of DNA from bacterial fractions extracted from the organic soil Seim and the mineral soil Hau revealed similar complexity of the communities with 5700 and 4900 different bacterial genomes (g soil [dry wt])-1, respectively. Thermal denaturation studies showed wide % G + C distributions in DNA from bacteria of both soils. Differences in the median % G + C with 55 to 61% for the bacterial community in soil Seim and 61 to 66% for that in soil Hau indicated a higher proportion of bacteria with a high DNA G + C content in soil Hau. In situ hybridization with fluorescent (Cy3-labeled) probes targeting larger phylogenetic groups showed minor differences between both soils, and between direct detection of bacteria in dispersed soil slurries and in bacterial fractions extracted from soils through about 90% of the total bacteria were lost during extraction. In dispersed slurries of both soils, only probes ALF1b, SRB385, and PLA46 hybridized to cells accounting for more than 1% of the DAPI-stained cells, while numbers obtained after hybridization with probes ARCH915, BET42a, GAM42a, HGC69a, and CF319a were below the detection limit set at < 1%. These results were confirmed by in situ hybridization with horseradish peroxidase (HRP)-labeled probes and subsequent Cy3-tyramide signal amplification. In contrast, dot blot hybridization with probe HGC69a indicated significant amounts of Gram-positive bacteria with a high DNA G + C content in both soils. These could subsequently be visualized in non-dispersed soil slurries by in situ hybridization with HRP-labeled probe HGC69a and Cy3-tyramide signal amplification. Filamentous Gram-positive bacteria with a high DNA G + C content, likely actinomycetes, which are present in soil Hau in significant numbers are obviously destroyed by procedures used for soil dispersion.

Transfer in Marine Sediments of the Naturally Occurring Plasmid pRAS1 Encoding Multiple Antibiotic Resistance.

The results of microcosm experiments performed with the fish-pathogenic bacterium Aeromonas salmonicida acting as a donor showed that promiscuous plasmid pRAS1, which encodes tetracycline resistance, is transferred at a high frequency in marine sediments even in the absence of a selective factor. The presence of oxytetracycline resulted in an increase in the transfer frequency compared with that of a microcosm to which no selective factor was added. Transfer frequencies of 3.4 x 10 transconjugant per recipient and 3.6 transconjugants per donor cell were obtained in a microcosm to which oxytetracycline had been added. Hybridization with a DNA probe specific for plasmid pRAS1 revealed that 45.8% of the oxytetracycline-resistant isolates obtained from a microcosm with no selective pressure carried the plasmid, while 86.8% of the isolates obtained from a microcosm to which oxytetracycline had been added carried the plasmid. Phenotypic characterization of the transconjugants revealed that the plasmid had been transferred to a variety of different biotypes in both microcosms. The diversity among the transconjugants isolated from the microcosm to which oxytetracycline had been added was substantially lower than the diversity among the transconjugants isolated from the microcosm to which no selective agent had been added.

Distribution of tetracycline resistance determinants among gram-negative bacteria isolated from polluted and unpolluted marine sediments.

Tetracycline-resistant gram-negative bacteria were isolated from four different marine sediments in Scandinavia and analyzed with DNA probes for the determinant classes A to E. Colony hybridizations of 429 isolates revealed that class E is the dominating resistance determinant in these marine sediments. Comparison of fecally polluted and unpolluted sediments showed few determinant classes in unpolluted sediment and a complex composition of several determinant classes in polluted sediment. Total DNA extraction and analysis with DNA probes for determinant classes A to E resulted in no hybridization signal, because of the low number of gram-negative tetracycline-resistant bacteria. Identification of class E isolates revealed that this determinant is present not only in Aeromonas hydrophila, Escherichia coli, and Vibrio salmonicida but also in additional strains.