Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition

Serial analysis of ribosomal sequence tags (SARST) is a novel technique for characterizing microbial community composition. The SARST method captures sequence information from concatemers of short 16S rDNA polymerase chain reaction (PCR) amplicons from complex populations of DNA. Here, we describe a similar method, serial analysis of V6 ribosomal sequence tags (SARST-V6), which targets the V6 hypervariable region of bacterial 16S rRNA genes. The SARST-V6 technique exploits internal primer sequences to generate compatible restriction digest overhangs, thereby improving upon the efficiency of SARST. Serial analysis of V6 ribosomal sequence tags of bacterial community composition in hydrothermal marine sediments from Guaymas Basin resembled results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identified the same major bacterial groups, but only SARST-V6 recovered thermodesulfobacteria and gamma-proteobacteria sequences, while only full-length PCR product cloning recovered candidate division OP11 se-quences. There were differences in the relative frequencies of some phylotypes. The disparities reflect differences in the amplicon pool obtained during initial amplification that may result from different primer affinities or DNA degradation. These results demonstrate the utility of SARST-V6 in collecting taxonomically informative data for high-throughput analysis of microbial communities.     

Serial analysis of ribosomal sequence tags (SARST): a high-throughput method for profiling complex microbial communities

Two decades of culture-independent studies have confirmed that microbial communities represent the most complex and concentrated pool of phylogenetic diversity on the planet. There remains a need for innovative molecular tools that can further our knowledge of microbial diversity and its functional implications. We present the method and application of serial analysis of ribosomal sequence tags (SARST) as a novel tool for elucidating complex microbial communities, such as those found in soils and sediments. Serial analysis of ribosomal sequence tags uses a series of enzymatic reactions to amplify and ligate ribosomal sequence tags (RSTs) from bacterial small subunit rRNA gene (SSU rDNA) V1-regions into concatemers that are cloned and sequenced. This approach offers a significant increase in throughput over traditional SSU rDNA clone libraries, as up to 20 RSTs are obtained from each sequencing reaction. To test SARST and measure the bias associated with this approach, RST libraries were prepared from a defined mixture of pure cultures and from duplicate arctic soil DNA samples. The actual RST distribution reflected the theoretical composition of the original defined mixture. Data from duplicate soil libraries (1345 and 1217 RSTs, with 525 and 505 unique RSTs, respectively) indicated that replication provides a strongly correlated RST profile (r(2) = 0.80) and division-level distribution of RSTs (r(2) = 0.99). Using sequence data from abundant soil RSTs, we designed specific primers that successfully amplified a larger portion of the SSU rDNA for further phylogenetic analysis. These results suggest that SARST is a powerful approach for reproducible high-throughput profiling of microbial diversity amenable to medical, industrial or environmental microbiology applications.     

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.     

Composition of microbial communities in hexachlorocyclohexane (HCH) contaminated soils from Spain revealed with a habitat-specific microarray

Microarray technology was used to characterize and compare hexachlorocyclohexane (HCH) contaminated soils from Spain. A library of 2,290 hypervariable 16S rRNA gene sequences was prepared with serial analysis of ribosomal sequence tags (SARST) from a composite of contaminated and uncontaminated soils. By designing hybridization probes specific to the 100 most abundant ribosomal sequence tags (RSTs) in the composite library, the RST array was designed to be habitat-specific and predicted to monitor the most abundant polymerase chain reaction (PCR)-amplified phylotypes in the individual samples. The sensitivity and specificity of the RST array was tested with a series of pure culture-specific probes and hybridized with labelled soil PCR products to generate hybridization patterns for each soil. Sequencing of prominent bands in denaturing gradient gel electrophoresis (DGGE) fingerprints derived from these soils provided a means by which we successfully confirmed the habitat-specific array design and validated the bulk of the probe signals. Non-metric multidimensional scaling revealed correlations between probe signals and soil physicochemical parameters. Among the strongest correlations to total HCH contamination were probe signals corresponding to unknown Gamma Proteobacteria, potential pollutant-degrading phylotypes, and several organisms with acid-tolerant phenotypes. The strongest correlations to alpha-HCH were probe signals corresponding to the genus Sphingomonas, which contains known HCH degraders. This suggests that the population detected was enriched in situ by HCH contamination and may play a role in HCH degradation. Other environmental parameters were also likely instrumental in shaping community composition in these soils. The results highlight the power of habitat-specific microarrays for comparing complex microbial communities.     

Analysis of the Rumen Bacterial Diversity under two Different Diet Conditions using Denaturing Gradient Gel Electrophoresis,Random Sequencing,and Statistical Ecology Approaches

Bacterial community structure and diversity in the rumen of steers in conditions of hay and corn diets was assessed by in vitro retrieval and analysis of the variable region (V3) of 16S rDNA. Two types of libraries were generated in this study: DGGE libraries, which further were analysed by excising, reamplification, and sequencing, and random shotgun sequence libraries. Phylogenetic and sequence similarity analyses of the resultant 68 clone sequences in DGGE libraries revealed the presence of 42 operational taxonomic units (OTUs) or phylotypes defined as having more than 97% of sequence similarity. One hundred and thirty four clone sequences in shotgun libraries were clustered into 72 phylotypes. The phylotype similarity, diversity, richness, and evenness in these libraries were estimated using a variety of diversity indices. In relation to diet, the corn-fed animals displayed more diverse and rich bacterial populations, which were mostly contributed by CFB-related phylotypes. Proteobacteria were also numerically prevalent on this diet (27%) but were represented by a few phylotypes thus diminishing the overall diversity and species richness values. On hay diet, the principal contributors to general diversity and species richness appeared to be low-G + C gram-positives. Although the ruminal Treponemaes were encountered only in hay-fed animals, their impact on species diversity on hay diet was low because of the limited number of phylotypes.     

Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of > 0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G + C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria ( Desulfosarcina group, Syntrophus and Geobacter / Pelobacter / Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.     

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Three methanogen 16S rRNA gene clone libraries were constructed from liquid (LM), solid (SM) and epithelium (EM) fractions taken from the rumen of Jinnan cattle in China. After the amplification by PCR using methanogen-specific primers Met86F and Met1340R, equal quantities of PCR products from the same fractions from each of the four cattle were mixed together and used to construct the three libraries. Sequence analysis showed that the 268 LM clones were divided into 35 phylotypes with 18 sequences of phylotypes affiliated with the genus Methanobrevibacter (84.3% of clones). The 135 SM clones were divided into 19 phylotypes with 11 phylotypes affiliated with the genus Methanobrevibacter (77.8%). The 267 EM clones were divided into 33 phylotypes with 15 phylotypes affiliated with the genus Methanobrevibacter (77.2%). Clones closely related to Methanomicrobium mobile and Methanobrevibacter wolinii were only found in the LM library, and those to Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii only in the SM library. LM library comprised 12.4% unidentified euryarchaeal clones, SM library 23.7% and EM library 25.5%, respectively. Five phylotypes (accession number: EF055528 and EF055531-EF055534) did not belong to the Euryarchaeota sequences we had known. One possible new genus (represented by phylotype E17, accession number EF055528) belonging to Methanobacteriaceae was identified from EM library. Quantitative real-time PCR for the first time revealed that epithelium fraction had significantly higher density of methanogens, with methanogenic mcrA gene copies (9.95 log 10 (copies per gram of wet weight)) than solid (9.26, P < 0.01) and the liquid (8.44, P < 0.001). The three clone libraries also appeared different in Shannon index (EM library 2.12, LM library 2.05 and SM library 1.73). Our results showed that there were apparent differences in the methanogenic diversity and abundance in the three different fractions within the rumen of Jinnan cattle, with Methanobrevibacter species predominant in all the three libraries and with epithelium fraction having more unknown species and higher density of methanogens.     

Characterization of the Dominant and Rare Members of a Young Hawaiian Soil Bacterial Community with Small-Subunit Ribosomal DNA Amplified from DNA Fractionated on the Basis of Its Guanine and Cytosine Composition

The small-subunit ribosomal DNA (rDNA) diversity was found to be very high in a Hawaiian soil community that might be expected to have lower diversity than the communities in continental soils because the Hawaiian soil is geographically isolated and only 200 years old, is subjected to a constant climate, and harbors low plant diversity. Since an underlying community structure could not be revealed by analyzing the total eubacterial rDNA, we first fractionated the DNA on the basis of guanine-plus-cytosine (G+C) content by using bis-benzimidazole and equilibrium centrifugation and then analyzed the bacterial rDNA amplified from a fraction with a high biomass (63% G+C fraction) and a fraction with a low biomass (35% G+C fraction). The rDNA clone libraries were screened by amplified rDNA restriction analysis to determine phylotype distribution. The dominant biomass reflected by the 63% G+C fraction contained several dominant phylotypes, while the community members that were less successful (35% G+C fraction) did not show dominance but there was a very high diversity of phylotypes. Nucleotide sequence analysis revealed taxa belonging to the groups expected for the G+C contents used. The dominant phylotypes in the 63% G+C fraction were members of the Pseudomonas, Rhizobium-Agrobacterium, and Rhodospirillum assemblages, while all of the clones sequenced from the 35% G+C fraction were affiliated with several Clostridium assemblages. The two-step rDNA analysis used here uncovered more diversity than can be detected by direct rDNA analysis of total community DNA. The G+C separation step is also a way to detect some of the less dominant organisms in a community.     

Analysis of Stomach and Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) from Coastal Louisiana,USA

We used high throughput pyrosequencing to characterize stomach and gut content microbiomes of Crassostrea virginica, the Easter oyster, obtained from two sites, one in Barataria Bay (Hackberry Bay) and the other in Terrebonne Bay (Lake Caillou), Louisiana, USA. Stomach microbiomes in oysters from Hackberry Bay were overwhelmingly dominated by Mollicutes most closely related to Mycoplasma; a more rich community dominated by Planctomyctes occurred in Lake Caillou oyster stomachs. Gut communities for oysters from both sites differed from stomach communities, and harbored a relatively diverse assemblage of phylotypes. Phylotypes most closely related to Shewanella and a Chloroflexi strain dominated the Lake Caillou and Hackberry Bay gut microbiota, respectively. While many members of the stomach and gut microbiomes appeared to be transients or opportunists, a putative core microbiome was identified based on phylotypes that occurred in all stomach or gut samples only. The putative core stomach microbiome comprised 5 OTUs in 3 phyla, while the putative core gut microbiome contained 44 OTUs in 12 phyla. These results collectively revealed novel microbial communities within the oyster digestive system, the functions of the oyster microbiome are largely unknown. A comparison of microbiomes from Louisiana oysters with bacterial communities reported for other marine invertebrates and fish indicated that molluscan microbiomes were more similar to each other than to microbiomes of polychaetes, decapods and fish.     

Diversity within cyanobacterial mat communities in variable salinity meltwater ponds of McMurdo Ice Shelf, Antarctica

This study investigated the diversity of cyanobacterial mat communities of three meltwater ponds--Fresh, Orange and Salt Ponds, south of Bratina Island, McMurdo Ice Shelf, Antarctica. A combined morphological and genetic approach using clone libraries was used to investigate the influence of salinity on cyanobacterial diversity within these ecosystems without prior cultivation or isolation of cyanobacteria. We were able to identify 22 phylotypes belonging to Phormidium sp., Oscillatoria sp. and Lyngbya sp. In addition, we identified Antarctic Nostoc sp., Nodularia sp. and Anabaena sp. from the clone libraries. Fresh (17 phylotypes) and Orange (nine phylotypes) Ponds showed a similar diversity in contrast to that of the hypersaline Salt Pond (five phylotypes), where the diversity within cyanobacterial mats was reduced. Using the comparison of identified phylotypes with existing Antarctic sequence data, it was possible to gain further insight into the different levels of distribution of phylotypes identified in the investigated cyanobacterial mat communities of McMurdo Ice Shelf.     

Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L.

The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated. A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA. The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation. The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library. The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48%. Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring. The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated. The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes. The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community. The diversity within the archaeal domain was low. Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity). The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea. The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.     

Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the Bacteroides and Prevotella group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the Mycoplasma without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.     

Molecular characterization of polychlorinated biphenyl-dechlorinating populations in contaminated sediments

Polychlorinated biphenyl (PCB)-dechlorinating microorganisms were characterized in PCB-contaminated sediments using amplified ribosomal DNA restriction analysis (ARDRA). The sediments were prepared by spiking Aroclor 1248 into PCB-free sediments, and were inoculated with microorganisms eluted from St. Lawrence River sediments. PCB-free sediments inoculated with the same inoculum served as the control. Four restriction fragment length polymorphism (RFLP) groups in the eubacterial and two in the archaeal domain were found exclusively in PCB-spiked sediment clone libraries. Sequence analysis of the four eubacterial clones showed homology to Escherichia coli, Lactosphaera pasteurii, Clostridium thermocellum, and Dehalobacter restrictus. The predominant archaeal sequence in the PCB-spiked sediment clone library was closely related to Methanosarcina barkeri, which appear to support earlier findings that methanogens are involved in PCB dechlorination. When the dot-blot hybridization was performed between the sediment DNA extract and the probes designed with eubacterial RFLP groups, the intensity of two of eubacterial RFLP groups, which showed high sequence homology to C. pascui and D. restrictus, was highly correlated with the number of dechlorinating microorganisms suggesting these two members intend to contribute to PCB dechlorination.     

Prokaryotic diversity in continuous cropping and rotational cropping soybean soil

In spite of the techniques based on the amplification of 16S rRNA genes (16S rDNA) to compare bacterial communities that are now widely in use in microbial ecology, little is known about the composition of the soybean continuous cropping (CC) and rotational cropping (RC) soil microbial community. To address this, we compared the levels of bacterial community diversity in RC and 5-year CC rhizosphere soil samples. We selected 407 clones in RC and 490 clones in CC for restriction fragment length polymorphism analysis. A total of 123 phylotypes were identified among the 16S rDNA clones, while 78 unique and 21 common phylotypes were identified among the CC soil isolates. Analysis of sequences from a subset of the phylotypes showed that at least 11 bacterial divisions were represented in the clone libraries. The phylotype richness, frequency distribution (evenness), and composition of the two clone libraries were investigated using a variety of diversity indices. Although the analysis of diversity indices and LIBSHUFF comparisons revealed that the compared libraries were not significantly different ( P =0.05) between the RC vs. CC soils, some differences could be observed in terms of specific phyla and groups. We concluded that the group variance was not determined immediately by the cropping system's induction, but was a long-term and slow process.     

Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Bacterial Community Composition in Different Sediments from the Eastern Mediterranean Sea: a Comparison of Four 16S Ribosomal DNA Clone Libraries

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.     

Biodiversity, Community Structural Shifts, and Biogeography of Prokaryotes within Antarctic Continental Shelf Sediment

16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region. Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions). The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs). At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes. At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria. Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core. Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment.     

Molecular diversity of methanogens in feedlot cattle from Ontario and Prince Edward Island, Canada

The molecular diversity of rumen methanogens in feedlot cattle and the composition of the methanogen populations in these animals from two geographic locations were investigated using 16S rRNA gene libraries prepared from pooled PCR products from 10 animals in Ontario (127 clones) and 10 animals from Prince Edward Island (114 clones). A total of 241 clones were examined, with Methanobrevibacter ruminantium accounting for more than one-third (85 clones) of the clones identified. From these 241 clones, 23 different 16S rRNA phylotypes were identified. Feedlot cattle from Ontario, which were fed a corn-based diet, revealed 11 phylotypes (38 clones) not found in feedlot cattle from Prince Edward Island, whereas the Prince Edward Island cattle, which were fed potato by-products as a finishing diet, had 7 phylotypes (42 clones) not found in cattle from Ontario. Five sequences, representing the remaining 161 clones (67% of the clones), were common in both herds. Of the 23 different sequences, 10 sequences (136 clones) were 89.8 to 100% similar to those from cultivated methanogens belonging to the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales, and the remaining 13 sequences (105 clones) were 74.1 to 75.8% similar to those from Thermoplasma volcanium and Thermoplasma acidophilum. Overall, nine possible new species were identified from the two clone libraries, including two new species belonging to the order Methanobacteriales and a new genus/species within the order Methanosarcinales. From the present survey, it is difficult to conclude whether the geographical isolation between these two herds or differences between the two finishing diets directly influenced community structure in the rumen. Further studies are warranted to properly assess the differences between these two finishing diets.