A comparison of methods for counting viruses in aquatic systems.
(17/614)
In this study, we compared different methods-including transmission electron microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4',6'-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1, 3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10(8) particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids. (+info)
Assessment of oceanic productivity with the triple-isotope composition of dissolved oxygen.
(18/614)
Plant production in the sea is a primary mechanism of global oxygen formation and carbon fixation. For this reason, and also because the ocean is a major sink for fossil fuel carbon dioxide, much attention has been given to estimating marine primary production. Here, we describe an approach for estimating production of photosynthetic oxygen, based on the isotopic composition of dissolved oxygen of seawater. This method allows the estimation of integrated oceanic productivity on a time scale of weeks. (+info)
Culturability and In situ abundance of pelagic bacteria from the North Sea.
(19/614)
The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton. (+info)
Biofilm community structure in polluted rivers: abundance of dominant phylogenetic groups over a complete annual cycle.
(20/614)
The seasonal dynamics of river biofilm communities in two German rivers, the Elbe and one of its tributaries, the Spittelwasser, were investigated for the first time by using fluorescence in situ hybridization and a standardized biofilm sampling procedure. We show the importance of members of the beta subclass of the class Proteobacteria, which formed the largest single group in the massively polluted Spittelwasser at all times. Clear seasonal peaks of abundance were observed for the planctomycetes and the Cytophaga-Flavobacterium cluster. (+info)
Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.
(21/614)
A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-microm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes. (+info)
Isotopic evidence for variations in the marine calcium cycle over the Cenozoic.
(22/614)
Significant variations in the isotopic composition of marine calcium have occurred over the last 80 million years. These variations reflect deviations in the balance between inputs of calcium to the ocean from weathering and outputs due to carbonate sedimentation, processes that are important in controlling the concentration of carbon dioxide in the atmosphere and, hence, global climate. The calcium isotopic ratio of paleo-seawater is an indicator of past changes in atmospheric carbon dioxide when coupled with determinations of paleo-pH. (+info)
Impacts of climatic change and fishing on Pacific salmon abundance over the past 300 years.
(23/614)
The effects of climate variability on Pacific salmon abundance are uncertain because historical records are short and are complicated by commercial harvesting and habitat alteration. We use lake sediment records of delta15N and biological indicators to reconstruct sockeye salmon abundance in the Bristol Bay and Kodiak Island regions of Alaska over the past 300 years. Marked shifts in populations occurred over decades during this period, and some pronounced changes appear to be related to climatic change. Variations in salmon returns due to climate or harvesting can have strong impacts on sockeye nursery lake productivity in systems where adult salmon carcasses are important nutrient sources. (+info)
Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts.
(24/614)
Enrichment experiments with North Sea bacterioplankton were performed to test if rapid incubation-induced changes in community structure explain the frequent isolation of members of a few particular bacterial lineages or if readily culturable bacteria are common in the plankton but in a state of dormancy. A metabolic inhibitor of cell division (nalidixic acid [NA]) was added to substrate-amended (S+) and unamended (S-) grazer-free seawater samples, and shifts in community composition and per cell DNA and protein content were compared with untreated controls. In addition, starvation survival experiments were performed on selected isolates. Incubations resulted in rapid community shifts towards typical culturable genera rather than in the activation of either dormant cells or the original DNA-rich bacterial fraction. Vibrio spp. and members of the Alteromonas/Colwellia cluster (A/C) were selectively enriched in S+ and S-, respectively, and this trend was even magnified by the addition of NA. These increases corresponded with the rise of cell populations with distinctively different but generally higher protein and DNA content in the various treatments. Uncultured dominant gamma-proteobacteria affiliating with the SAR86 cluster and members of the culturable genus Oceanospirillum were not enriched or activated, but there was no indication of substrate-induced cell death, either. Strains of Vibrio and A/C maintained high ribosome levels in pure cultures during extended periods of starvation, whereas Oceanospirillum spp. did not. The life strategy of rapidly enriched culturable gamma-proteobacteria could thus be described as a "feast and famine" existence involving different activation levels of substrate concentration. (+info)