Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).     

Microarray and Functional Gene Analyses of Sulfate-Reducing Prokaryotes in Low-Sulfate, Acidic Fens Reveal Cooccurrence of Recognized Genera and Novel Lineages

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 μM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family “Syntrophobacteraceae” were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.     

Unexpected population distribution in a microbial mat community: sulfate-reducing bacteria localized to the highly oxic chemocline in contrast to a eukaryotic preference for anoxia.

The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-microm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families "Desulfovibrionaceae" and "Desulfobacteriaceae," Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia.     

Biogeography of sulfate-reducing prokaryotes in river floodplains

In this study, a large-scale field survey was conducted to describe the biogeography of sulfate-reducing prokaryotes (SRPs) in river floodplains. Fingerprints obtained with three methods, i.e. 16S rRNA gene-based oligonucleotide microarray, dsrB-based denaturing gradient gel electrophoresis (DGGE) and polar lipid-derived fatty acid (PLFA) analyses, were used as a proxy to describe the SRPs community diversity. Each set of profiles was subjected to a combined multivariate/correlation analysis in order to compare SRP community profiles and to highlight the environmental variables influencing the SRPs distribution along environmental gradients. Floodplain soils harbored distinct SRP communities displaying biogeographic patterns. Nearly all profiles from the tidal sites consistently separated from the nontidal sites, independently from the screening method and the multivariate statistics used. The distribution of the microarray/DGGE/PLFA-based fingerprints in the principal component plots could be correlated to eight soil variables, i.e. soil organic matter, total nitrogen, total phosphorous and total potassium, and extractable ammonium, nitrate, phosphate and sulfate, as well as seven pore water variables, i.e. phosphate, sulfate, sulfide, chloride, sodium, potassium and magnesium ions. Indication of a salinity- and plant nutrient-dependent distribution of SRPs related to Desulfosarcina, Desulfomonile and Desulfobacter was suggested by microarray, DGGE and PLFA analyses.     

Vertical distribution and diversity of sulfate-reducing prokaryotes in the Pearl River estuarine sediments, Southern China

The vertical distribution and diversity of sulfate-reducing prokaryotes (SRPs) in a sediment core from the Pearl River Estuary was reported for the first time. The profiles of methane and sulfate concentrations along the sediment core indicated processes of methane production/oxidation and sulfate reduction. Phospholipid fatty acids analysis suggested that sulfur-oxidizing bacteria (SOB) might be abundant in the upper layers, while SRPs might be distributed throughout the sediment core. Quantitative competitive-PCR analysis indicated that the ratios of SRPs to total bacteria in the sediment core varied from around 2–20%. Four dissimilatory sulfite reductase ( dsrAB) gene libraries were constructed and analyzed for the top layer (0–6 cm), middle layer (18–24 cm), bottom layer (44–50 cm) and the sulfate-methane transition zone (32–42 cm) sediments. Most of the retrieved dsrAB sequences (80.9%) had low sequence similarity with known SRP sequences and formed deeply branching dsrAB lineages. Meanwhile, bacterial 16S rRNA gene analysis revealed that members of the Proteobacteria were predominant in these sediments. Putative SRPs within Desulfobacteriaceae, Syntrophaceae and Desulfobulbaceae of Deltaproteobacteria , and putative SOB within Epsilonproteobacteria were detected by the 16S rRNA gene analysis. Results of this study suggested a variety of novel SRPs in the Pearl River Estuary sediments.     

Unexpected Population Distribution in a Microbial Mat Community: Sulfate-Reducing Bacteria Localized to the Highly Oxic Chemocline in Contrast to a Eukaryotic Preference for Anoxia

The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-μm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families “Desulfovibrionaceae” and “Desulfobacteriaceae,” Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia.     

Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA

An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.     

Quantification of microbial communities in near-surface and deeply buried marine sediments on the Peru continental margin using real-time PCR

Deeply buried marine sediments harbour a large fraction of all prokaryotes on Earth but it is still unknown which phylogenetic and physiological microbial groups dominate the deep biosphere. In this study real-time PCR allowed a comparative quantitative microbial community analysis in near-surface and deeply buried marine sediments from the Peru continental margin. The 16S rRNA gene copy numbers of prokaryotes and Bacteria were almost identical with a maximum of 10(8)-10(10) copies cm(-3) in the near-surface sediments. Archaea exhibited one to three orders of magnitude lower 16S rRNA gene copy numbers. The 18S rRNA gene of Eukarya was always at least three orders of magnitude less abundant than the 16S rRNA gene of prokaryotes. The 16S rRNA gene of the Fe(III)- and Mn(IV)-reducing bacterial family Geobacteraceae and the dissimilatory (bi)sulfite reductase gene (dsrA) of sulfate-reducing prokaryotes were abundant with 10(6)-10(8) copies cm(-3) in near-surface sediments but showed lower numbers and an irregular distribution in the deep sediments. The copy numbers of all genes decreased with sediment depth exponentially. The depth gradients were steeper for the gene copy numbers than for numbers of total prokaryotes (acridine orange direct counts), which reflects the ongoing degradation of the high-molecular-weight DNA with sediment age and depth. The occurrence of eukaryotic DNA also suggests DNA preservation in the deeply buried sediments.     

Archaeal Microbial Diversity of Hypersaline Lake Acıgöl,Denizli, Turkey

Archaeal diversity in Lake Ac?göl, a closed-basin, alkaline, hypersaline lake located at the northern edge of western Tourides in southwest Anatolia, was investigated using culture-independent methods. Microbial mat samples were collected from six different points. Archaeal 16S rRNA gene libraries were generated using domain specific oligonucleotide primers, and 16S rRNA gene sequences of clone libraries were analyzed phylogenetically. Denaturing gradient gel electrophoresis of 16S rRNA genes showed a variance in diversity with spatial differences. Archaeal diversity of Ac?göl is dominated by the members of family Halobacteriaceae which requires both high salt concentration and high pH for growth. Sequence analysis of archaeal 16s rRNA genes indicates the presence of the phylotypes affiliated with the genera Halorubrum, Halosimplex, Halorhabdus, Haloterrigena and Natronococcus in the analyzed samples.     

Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments.

The sulfate-reducing bacterial populations of a stratified marine water column, Mariager Fjord, Denmark, were investigated by molecular and culture-dependent approaches in parallel. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA and DNA encoding rRNA (rDNA) isolated from the water column indicated specific bacterial populations in different water column layers and revealed a highly differentiated pattern of rRNA- and rDNA-derived PCR amplificates, probably reflecting active and resting bacterial populations. Hybridization of DGGE patterns with rRNA probes indicated the increased presence and activity (by at least 1 order of magnitude) of sulfate-reducing bacteria within and below the chemocline. Parallel to this molecular approach, an approach involving most-probable-number (MPN) counts was used, and it found a similar distribution of cultivable sulfate-reducing bacteria in the water column of Mariager Fjord, Approximately 25 cells and 250 cells per ml above and below the chemocline, respectively, were found. Desulfovibrio- and Desulfobulbus-related strains occurred in the oxic zone. DGGE bands from MPN cultures were sequenced and compared with those obtained from nucleic acids extracted from water column samples. The MPN isolates were phylogenetically affiliated with sulfate-reducing delta subdivision proteobacteria (members of the genera Desulfovibrio, Desulfobulbus, and Desulfobacter), whereas the molecular isolates constituted an independent lineage of the delta subdivision proteobacteria. DGGE of PCR-amplified nucleic acids with general eubacterial PCR primers conceptually revealed the general bacterial population, whereas the use of culture media allowed cultivable sulfate-reducing bacteria to be selected. A parallel study of Mariager Fjord biogeochemistry, bacterial activity, and bacterial counts complementing this investigation has been presented elsewhere (N.B. Ramsing, H. Fossing, T. G. Ferdelman, F. Andersen, and B. Thamdrup, Appl. Environ.     

Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.     

In situ analysis of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland)

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) revealed the presence of a diverse number of phototrophic sulfur bacteria. Sequences resembled those of rRNA of type strains Chromatium okenii DSM169 and Amoebobacter purpureus DSM4197, as well as those of four bacteria forming a tight cluster with A. purpureus DSM4197 and Lamprocystis roseopersicina DSM229. In situ hybridization with fluorescent (Cy3 labeled) oligonucleotide probes indicated that all large-celled phototrophic sulfur bacteria in the chemocline of Lake Cadagno were represented by C. okenii DSM169, while small-celled phototrophic sulfur bacteria consisted of four major populations with different distribution profiles in the chemocline indicating different ecophysiological adaptations.     

In situ analysis of sulfate-reducing bacteria related to Desulfocapsa thiozymogenes in the chemocline of meromictic Lake Cadagno (Switzerland)

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) retrieved two clusters of sequences resembling sulfate-reducing bacteria within the family Desulfovibrionaceae. In situ hybridization showed that, similar to sulfate-reducing bacteria of the family Desulfobacteriaceae, bacteria of one cluster with similarity values to the closest cultured relatives of between 92.6 and 93.1% resembled free cells or cells loosely attached to other cells or debris. Bacteria of the second cluster closely related to Desulfocapsa thiozymogenes DSM7269 with similarity values between 97. 9 and 98.4% were generally associated with aggregates of different small-celled phototrophic sulfur bacteria, suggesting a potential interaction between the two groups of bacteria.     

Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization

Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.     

Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.     

Oligonucleotide microarray for identification of Enterococcus species

For detection of most members of the Enterococcaceae, the specificity of a novel oligonucleotide microarray (ECC-PhyloChip) consisting of 41 hierarchically nested 16S or 23S rRNA gene-targeted probes was evaluated with 23 pure cultures (including 19 Enterococcus species). Target nucleic acids were prepared by PCR amplification of a 4.5-kb DNA fragment containing large parts of the 16S and 23S rRNA genes and were subsequently labeled fluorescently by random priming. Each tested member of the Enterococcaceae was correctly identified on the basis of its unique microarray hybridization pattern. The evaluated ECC-PhyloChip was successfully applied for identification of Enterococcus faecium and Enterococcus faecalis in artificially contaminated milk samples demonstrating the utility of the ECC-PhyloChip for parallel identification and differentiation of Enterococcus species in food samples.     

Diversity and abundance of uncultured cytophaga-like bacteria in the Delaware estuary

The Cytophaga-Flavobacterium group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material. However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes. To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the Cytophaga-Flavobacterium group and was used to construct a library of these genes from the Delaware Estuary. This library had several novel Cytophaga-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments. An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays. Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean). The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries. The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea. DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.     

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.