Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.Ribosomal RNA genes (rDNAs) are extensively used to study the diversity of microorganisms in environmental samples. To date, most surveys of microbial diversity in environmental samples have relied upon cloning and sequencing of rDNAs. These studies have shown that the diversity of microorganisms in natural ecosystems has been severely underestimated in culture collections and have led to the discovery of several new microbial lineages (5,10,20,28). Despite the increased knowledge of the phylogenetic diversity of indigenous microbes, less is known about the abundance of particular groups or their spatial and temporal dynamics.Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Recoveries of PCR-amplified rDNAs from clone libraries are subject to several quantitative biases (see reference 26 for a review). rRNAbased quantification therefore has relied mainly on radiolabeled oligonucleotide hybridization to extracted rRNA (11,16,21) or whole-cell hybridization using fluorescence-labeled oligonucleotides (1, 7). However, in many ecosystems the abundance of microorganisms is relatively low and many cells may have a low rRNA content, limiting the sensitivity of oligonucleotide hybridization techniques for quantification of specific microorganisms.In individual cells, the number of copies of rDNAs may be several orders of magnitude lower than that of rRNAs; in DNA, rRNA coding sequences exist in a context of much higher genetic complexity. It is therefore difficult to quantify rDNAs by oligonucleotide hybridization. However, rDNA is suitable for amplification by the PCR. Provided that the amplification efficienc...

The recent isolation of the ammonia-oxidizing crenarchaeon Nitrosopumilus maritimus has expanded the known phylogenetic distribution of nitrifying phenotypes beyond the domain Bacteria. To further characterize nitrification in the marine environment and explore the potential crenarchaeal contribution to this process, we quantified putative nitrifying genes and phylotypes in picoplankton genomic libraries and environmental DNA samples from coastal and open ocean habitats. Betaproteobacteria ammonia monooxygenase subunit A (amoA) gene copy numbers were low or undetectable, in stark contrast to crenarchaeal amoA-like genes that were broadly distributed and reached up to 6 x 10(4) copies ml(-1). Unexpectedly, in the North Pacific Subtropical Gyre, a deeply branching crenarchaeal group related to a hot spring clade (pSL12) was at times abundant below the euphotic zone. Quantitative data suggested that the pSL12 relatives also contain archaeal amoA-like genes. In both coastal and open ocean habitats, close relatives of known nitrite-oxidizing Nitrospina species were well represented in genomic DNA libraries and quantitative PCR profiles. Planktonic Nitrospina depth distributions correlated with those of Crenarchaea. Overall, the data suggest that amoA-containing Crenarchaea are more phylogenetically diverse than previously reported. Additionally, distributional patterns of planktonic Crenarchaea and Nitrospina species suggest potential metabolic interactions between these groups in the ocean's water column.

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant 13 C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia.13 C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of ␦-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong 13 C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant 13 C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90°C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H 2 , acetate, and CO 2 as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.Over the past decade, microbiological investigations of high temperature, petroleum-rich strata from a number of geographically distant sites have revealed physiologically diverse assemblages of thermophilic and hyperthermophilic anaerobic microorganisms. Physiological types isolated from these biotopes include sulfate reducers (54, 58), sulfidogens (31, 54), fermentative bacteria (12, 22), manganese and iron reducers (23), methanogens (42, 52), and acetogens (13). Despite the description of an increasing number of new thermophilic species, relatively little information is available on the composition of microbial assemblages in these unique subsurface environments. This is primarily due to the reliance on cultured-based methods for the recovery and identification of individual oil field isolates and the focus on specific physiological groups of microorganisms, such as sulfate-reducing and fermentative microorganisms, rather than the entire subsurface microbial community.Culture-based approaches, while extremely useful for understanding the physiological potential of isolated organisms, do not necessarily provide comprehensive information on the comp...

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is "Candidatus Arcobacter sulfidicus." For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO 2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO 2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by "Candidatus Arcobacter sulfidicus" or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.In the marine environment, hydrogen sulfide is a ubiquitous end product of anaerobic processes of organic matter remineralization (5,23,29,30). At ridge crest sites on the ocean floor, it is produced from the geothermal transformation of sulfate and elemental sulfur leaching via seawater-basaltic rock interaction (26,40,62). When brought into contact with the aerobic biosphere, hydrogen sulfide becomes an energy-yielding substrate for chemosynthetic colorless sulfur-oxidizing bacteria. Members of this group include free-living rods or ovoids of the genera Thiobacillus, Thiomonas, Acidiphilium, Thiomicrospira, and Thiovulum (31,32,34,42) as well as the morphologically conspicuous gliding and nongliding filamentous forms of the genera Beggiatoa, Thioploca, and Thiothrix (19,47,48,69). These organisms are characterized by their ability to catalyze the oxidation of sulfide and its chemically and biologically mediated partial oxidation products (polysulfides, S n 2Ϫ ; elemental sulfur, S 0 ; sulfane monosulfonic acids, HSS n O 3 2Ϫ ]; and polythionates, S n O 6 2Ϫ ) (14, 33, 66) coupled to the fixation of carbon dioxide to organic carbon by utilizing the same CalvinBassham-Benson cycle enzymes as those used by oxygenic phototrophs.Because of the differential rates of oxidation of hydrogen sulfide and derived oxidation products, intermediates may substantially accu...