A protocol for enumeration of aquatic viruses by epifluorescence microscopy using Anodisc™ 13 membranes

<p>Abstract</p> <p>Background</p> <p>Epifluorescence microscopy is a common method used to enumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has not been previously reported.</p> <p>Results</p> <p>Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators' laboratories from commercially available Swinnex^®filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ small pore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples.</p> <p>Conclusions</p> <p>The 13 mm Anodisc membranes are less costly and require smaller sample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.</p

Genome Sequences of Two Temperate Phages, ΦCB2047-A and ΦCB2047-C, Infecting Sulfitobacter sp. Strain 2047

We announce the complete genome sequences of two temperate Podoviridae,Sulfitobacter phages ΦCB2047-A and ΦCB2047-C, which infect Sulfitobacter sp. strain 2047, a member of the Roseobacter clade. This is the first report of temperate podophage infecting members of the Sulfitobacter genus of theRoseobacter clade

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis

Genome Sequence of the Sulfitobacter sp. Strain 2047-Infecting Lytic Phage ΦCB2047-B

We announce the complete genome sequence of a lytic podovirus, ΦCB2047-B, which infects the bacterium Sulfitobacter sp. strain 2047, a member of theRoseobacter clade. Genome analysis revealed ΦCB2047-B to be an N4-like phage, with its genome having high nucleotide similarity to other N4-like roseophage genomes

Relevance of ammonium oxidation within biological soil crust communities

Thin, vertically structured topsoil communities that become ecologically important in arid regions (biological soil crusts or BSCs) are responsible for much of the nitrogen inputs into pristine arid lands. We studied N2 fixation and ammonium oxidation (AO) at subcentimetre resolution within BSCs from the Colorado Plateau. Pools of dissolved porewater nitrate/nitrite, ammonium and organic nitrogen in wetted BSCs were high in comparison with those typical of aridosoils. They remained stable during incubations, indicating that input and output processes were of similar magnitude. Areal N2 fixation rates (6.5–48 µmol C2H2 m-2 h-1) were high, the vertical distribution of N2 fixation peaking close to the surface if populations of heterocystous cyanobacteria were present, but in the subsurface if they were absent. Areal AO rates (19–46 µmol N m-2 h-1) were commensurate with N2 fixation inputs. When considering oxygen availability, AO activity invariably peaked 2–3 mm deep and was limited by oxygen (not ammonium) supply. Most probable number (MPN)-enumerated ammonia-oxidizing bacteria (6.7–7.9 × 103 cells g−1 on average) clearly peaked at 2–3 mm depth. Thus, AO (hence nitrification) is a spatially restricted but important process in the nitrogen cycling of BSC, turning much of the biologically fixed nitrogen into oxidized forms, the fate of which remains to be determined

The influence of sample biases on estimations of marine microbial diversity

Background The Roseobacter clade is a widespread, metabolically versatile, and easily cultured lineage of marine microbes. Furthermore, cultured isolates are generally considered to be representative of the naturally occurring diversity in the environment. Roseobacter is thus considered an ideal clade for exploring and understanding microbial function in a representative model system. Nevertheless, recent studies question whether cultured strains are truly representative of natural populations. Support for one of these studies was based on the potentially biased samples of a Global Ocean Sampling (GOS) expedition [1]. The marine metagenomic studies of the GOS expeditions initially focused on microbes collected from a single filter range (0.1um – 0.8um) and it has been previously suggested that this excludes many Roseobacters, thus biasing the dataset towards smaller cells. Materials and methods Here, we take advantage of a GOS metagenomic dataset from a 2006-2008 Antarctic expedition that included community analyses over three different size classes (0.1um-0.8um; 0.8um-3um; and 3um-200um). We examined correlations between bacterial taxonomic diversity (based on 16S rRNA gene analyses) and the three size classes to understand the potential for diversity biases between filter ranges. The 16S rRNA gene sequences from these datasets were extracted using Metaxa and subsequently analyzed using MOTHUR, which provided fine-level resolution taxonomic assignments. Results This analysis reveals a potential bias as the ranges of Roseobacter subgroup diversity in this dataset varied between size classes. From this analysis we identified specific Roseobacter subgroups that were predominantly found in specific size classes. For instance, the OCT and Roseovarius subgroups were most prevalent in the 0.8um-3um filter range, while the DC5-80 and NAC11-7 subgroups were well represented in both 0.1um-0.8um and 0.8um-3um filter ranges. These findings reveal the value of analyzing multiple size class fractions when assessing microbial diversity