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Recent culture-based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy-based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60-150 per thousand) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100 per thousand salinity. The other isolates - Groups B-E - can all grow at 150-175 per thousand salinities and are probably moderate halophiles. Groups B-E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non-saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well-supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as 'MAST-13', which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.

The genetic diversity of picoplanktonic (i.e. cells that can pass through a 3 mum pore-size filter) green algae was investigated in the Mediterranean Sea in late summer by a culture-independent approach. Genetic libraries of the 18S rRNA gene were constructed using two different primer sets. The first set is commonly used to amplify the majority of eukaryotic lineages, while the second was composed of a general eukaryotic forward primer and a reverse primer biased towards the phylum Chloroplastida. A total of 3980 partial environmental sequences were obtained: 1668 using the general eukaryotic primer set and 2312 using the Chloroplastida-biased primer set. Of these sequences, 65 (4%) and 594 (26%) belonged to the Chloroplastida respectively. A 99.5% sequence similarity cut-off value allowed classification of these 659 Chloroplastida sequences into 74 different operational taxonomic units. A majority of the Chloroplastida sequences (99%) belonged to the prasinophytes. In addition to the seven independent prasinophyte lineages previously described, we discovered two new clades (clades VIII and IX), as well as a significant genetic diversity at the species and subspecies levels, notably among the genera Crustomastix, Dolichomastix and Mamiella (Mamiellales), but also within Pyramimonas and Halosphaera (Pyramimonadales). Such diversity within prasinophytes has not previously been observed by cloning approaches, illustrating the power of using targeted primers for clone library construction. Prasinophyte assemblages differed especially in relation to nutrient levels. Micromonas and Ostreococcus were mainly recovered from mesotrophic areas, whereas Mamiella, Crustomastix and Dolichomastix were mostly detected in oligotrophic surface waters. Within genera such as Ostreococcus or Crustomastix for which several clades were observed, depth seemed to be the main factor controlling differential distribution of genotypes.

Biotic communities and ecosystem dynamics in terrestrial Antarctica are limited by an array of extreme conditions including low temperatures, moisture and organic matter availability, high salinity, and a paucity of biodiversity to facilitate key ecological processes. Recent studies have discovered that the prokaryotic communities in these extreme systems are highly diverse with patchy distributions. Investigating the physical and biological controls over the distribution and activity of microbial biodiversity in Victoria Land is essential to understanding ecological functioning in this region. Currently, little information on the distribution, structure and activity of soil communities anywhere in Victoria Land are available, and their sensitivity to potential climate change remains largely unknown. We investigated soil microbial communities from low- and high-productivity habitats in an isolated Antarctic location to determine how the soil environment impacts microbial community composition and structure. The microbial communities in Luther Vale, Northern Victoria Land were analysed using bacterial 16S rRNA gene clone libraries and were related to soil geochemical parameters and classical morphological analysis of soil metazoan invertebrate communities. A total of 323 16S rRNA gene sequences analysed from four soils spanning a productivity gradient indicated a high diversity (Shannon-Weaver values > 3) of phylotypes within the clone libraries and distinct differences in community structure between the two soil productivity habitats linked to water and nutrient availability. In particular, members of the Deinococcus/Thermus lineage were found exclusively in the drier, low-productivity soils, while Gammaproteobacteria of the genus Xanthomonas were found exclusively in high-productivity soils. However, rarefaction curves indicated that these microbial habitats remain under-sampled. Our results add to the recent literature suggesting that there is a higher biodiversity within Antarctic soils than previously expected.

Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33 degrees 56'N, 102 degrees 52'E) region and under the annual temperature around 1 degrees C. Previous studies indicated that Zoige wetland was one of the CH(4) emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales, could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10(7) cells per gram of soil, accounting for about 30% of the total Archeae. By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO(2), methanol or trimethylamine was determined to increase to 10(8) cells ml(-1), but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30 degrees C and 15 degrees C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae, and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.

Samples of the sponge Haliclona simulans were collected from Irish waters and subjected to a culture-independent analysis to determine the microbial, polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) diversity. 16S rRNA gene libraries were prepared from total sponge, bacterial enriched sponge and seawater samples. Eight phyla from the Bacteria were detected in the sponge by phylogenetic analyses of the 16S rRNA gene libraries. The most abundant phylum in the total sponge library was the Proteobacteria (86%), with the majority of these clones being from the gamma-Proteobacteria (77%); two groups of clones were dominant and together made up 69% of the total. Both of these groups were related to other sponge-derived microbes and comprised novel genera. Within the other bacterial phyla groups of clones representing novel candidate genera within the phyla Verrucomicrobia and Lentisphaerae were also found. Selective enrichment of the bacterial component of the sponge prior to 16S rRNA gene analysis resulted in a 16S rRNA gene library dominated by a novel genus of delta-Proteobacteria, most closely related to the Bdellovibrio. The potential for the sponge microbiota to produce secondary metabolites was also analysed by polymerase chain reaction amplification of PKS and NRPS genes. While no NRPS sequences were isolated seven ketosynthase (KS) sequences were obtained from the sponge metagenome. Analyses of these clones revealed a diverse collection of PKS sequences which were most closely affiliated with PKS from members of the Cyanobacteria, Myxobacteria and Dinoflagellata.

Here, we have identified a protist (dinoflagellate) lineage that has diversified recently in evolutionary terms. The species members of this lineage inhabit cold-water marine and lacustrine habitats, which are distributed along a broad range of salinities (0-32) and geographic distances (0-18 000 km). Moreover, the species present different degrees of morphological and sometimes physiological variability. Altogether, we analysed 30 strains, generating 55 new DNA sequences. The nuclear ribosomal DNA (nrDNA) sequences (including rapidly evolving introns) were very similar or identical among all the analysed isolates. This very low nrDNA differentiation was contrasted by a relatively high cytochrome b (COB) mitochondrial DNA (mtDNA) polymorphism, even though the COB evolves very slowly in dinoflagellates. The 16 Maximum Likelihood and Bayesian phylogenies constructed using nr/mtDNA indicated that the studied cold-water dinoflagellates constitute a monophyletic group (supported also by the morphological analyses), which appears to be evolutionary related to marine-brackish and sometimes toxic Pfiesteria species. We conclude that the studied dinoflagellates belong to a lineage which has diversified recently and spread, sometimes over long distances, across low-temperature environments which differ markedly in ecology (marine versus lacustrine communities) and salinity. Probably, this evolutionary diversification was promoted by the variety of natural selection regimes encountered in the different environments.

The aim of this study was to explore the use of large-scale sequencing to better describe the genome content of naturally occurring, uncultured protists. We constructed a metagenomic fosmid library from a picoplanktonic assemblage (0.2-3 mum size cells) collected at the Blanes Bay Microbial Observatory (Western Mediterranean). Seven clones contained a small-subunit ribosomal RNA gene (SSU rDNA) affiliating with prasinophytes and uncultured alveolates. One clone (FBB25; 35 kb in size) was completely sequenced and found to be a tandem repeat array (5.5 times) of the rDNA operon, including three rRNA genes (SSU, large-subunit and 5.8S rDNAs) and three spacer regions (internal transcribed spacers 1, 2 and intergenic spacer). The SSU rDNA of FBB25 affiliated with the marine alveolates group I, cluster 1, and was almost identical to sequences retrieved only in marine surveys from a wide geographic and ecological range. Phylogenetic trees using the different rRNA genes showed FBB25 as an independent branch among the main alveolate groups, but their closest affiliation varied between the SSU tree (dinoflagellates) and the large-subunit and 5.8S trees (perkinsids). The spacer regions of FBB25 were particularly short when compared with other eukaryotes, indicating a possible genome streamlining in this picoeukaryote. Finally, not a single polymorphism was found in the rDNA repeat array, suggesting that the high SSU rDNA variability typically found in molecular surveys derives from organismal and not intragenomic diversity. This first report on the rDNA genomic structure of an uncultured marine alveolate improves their phylogenetic position and helps interpreting data generated during picoeukaryotic molecular surveys.

The bacterial bioaerosol community of eight swine confinement buildings (SCB) was monitored during two visits in the winter, and one during the summer. To our knowledge, culture-independent approaches and molecular biology tools such as biomass quantification and biodiversity analyses have never been applied to swine building bioaerosol analyses. Total DNA of each sample was extracted and analysed by quantitative real-time polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis using primers targeting the bacterial 16S rRNA gene. Even though the total bacterial concentration was higher in winter than in summer, the total bacterial concentration for both seasons was 100 to1000 times higher than the total cultural bacteria. The concentration of bioaerosol was influenced by the temperature indoors, which was regulated with an electronic fan system driving warm air and particles outside of the SCB. Comparison of the DGGE profiles showed the same biodiversity in each SCB during both seasons. The phylogenetic analysis revealed a large number of sequences (93.8%) related to Gram-positive anaerobic bacteria, such as Clostridia, and dominated by the Clostridia cluster I (C. disporicum) and the Clostridia cluster XI (C. glycolycum). The bioaerosol diversity also contained also a low proportion of Bacteroidetes and Lactobacillales-Streptococcales sequences. Analyses of the global community and phylotype diversity showed that the main source of bioaerosols could come from the pig manure slurry.

Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology.

Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchstädt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the 'cattle manure' itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore.

Insoluble plant polysaccharides and endogenous mucin are important energy sources for human colonic microorganisms. The object of this study was to determine whether or not specific communities colonize these substrates. Using faecal samples from four individuals as inocula for an anaerobic in vitro continuous flow system, the colonization of wheat bran, high amylose starch and porcine gastric mucin was examined. Recovered substrates were extensively washed and the remaining tightly attached bacterial communities were identified using polymerase chain reaction-amplified 16S rRNA gene sequences and fluorescent in situ hybridization. The substrate had a major influence on the species of attached bacteria detected. Sequences retrieved from bran were dominated by clostridial cluster XIVa bacteria, including uncultured relatives of Clostridium hathewayi, Eubacterium rectale and Roseburia species. Bacteroides species were also detected. The most abundant sequences recovered from starch were related to the cultured species Ruminococcus bromii, Bifidobacterium adolescentis, Bifidobacterium breve and E. rectale. The most commonly recovered sequences from mucin were from Bifidobacterium bifidum and uncultured bacteria related to Ruminococcus lactaris. This study suggests that a specific subset of bacteria is likely to be the primary colonizers of particular insoluble colonic substrates. For a given substrate, however, the primary colonizing species may vary between host individuals.

Deep-sea sediments of the eastern Mediterranean harbour a series of dark, organic carbon-rich layers, so-called sapropels. Within these layers, the carotenoid isorenieratene was detected. Since it is specific for the obligately anaerobic phototrophic green sulfur bacteria, the presence of isorenieratene may suggest that extended water column anoxia occurred in the ancient Mediterranean Sea during periods of sapropel formation. Only three carotenoids (isorenieratene, beta-isorenieratene and chlorobactene) are typical for green sulfur bacteria and thus do not permit to differentiate between the approximately 80 known phylotypes. In order to reconstruct the paleoecological conditions in more detail, we searched for fossil 16S rRNA gene sequences of green sulfur bacteria employing ancient DNA methodology. 540 bp-long fossil sequences could indeed be amplified from up to 217 000-year-old sapropels. In addition, such sequences were also recovered from carbon-lean intermediate sediment layers deposited during times of an entirely oxic water column. Unexpectedly, however, all the recovered 16S rRNA gene sequences grouped with freshwater or brackish, rather than truly marine, types of green sulfur bacteria. It is therefore feasible that the molecular remains of green sulfur bacteria originated from populations which thrived in adjacent freshwater or estuarine coastal environments rather than from an indigenous pelagic population.

Bacterial communities were examined in replicate lab-scale activated sludge reactors after a period of several months of enrichment with non-ionic nonylphenol ethoxylate (NPE) surfactants. Four sequential batch reactors were fed with synthetic sewage, two of which received additionally NPE. Small subunit rDNA-derived denaturing gel gradient electrophoresis (DGGE) profiles and 16S rDNA clone libraries were dominated by clones of Gammaproteobacteria class. Sequences of the other codominant rDNA phylotypes observed only in DGGE from NPE-amended reactors were, respectively, associated with the Group III of the Acidobacteria phylum. Intriguingly, 16S rRNA content from abundant Gammaproteobacteria cells was unexpectedly low. In addition to Acidobacteria, rRNA-derived DGGE profiles were dominated by members of the order Burkholderiales (of the Betaproteobacteria) and of the genus Sphingomonas (a member of the Alphaproteobacteria). Specific oligonucleotide probes for the selected ribotypes were designed and applied for quantitative real time polymerase chain reaction and fluorescence in situ hybridization, confirming their dominance in treated reactors. The parallel abundance of unique phylotypes in replicate reactors implies a distinctive selection of dominant organisms, which are better adapted to specialized niches in the highly selective environment.

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

We used slot blot hybridization, quantitative polymerase chain reaction (qPCR), and flow cytometry microarrays to quantify specific 16S rDNAs in weekly fecal specimens from four monkeys housed in a research vivarium for periods ranging from five to 8 months. Even in these uniformly housed and fed animals the gut microbiota is idiosyncratic, very dynamic on short timescales, and shows significant positive and negative correlations among some bacteria as well as responses to heavy metal exposure. The relative quantification (fmol targets per total fmol bacterial 16S rDNA) afforded by flow cytometry microarrays agreed well with the absolute quantification (nanogram of target DNA per nanogram of fecal DNA) afforded by slot blots and qPCR. We also noted strengths and weaknesses in inter-method comparisons for DNA-based quantification of these complex bacterial communities.

More effective control of membrane biofouling in membrane bioreactors (MBRs) lies in the fundamental understanding of the pioneer microorganisms responsible for surface colonization that leads to biofilm formation. In this study, the composition of the planktonic and sessile microbial communities inhabiting four laboratory-scale MBR systems were compared using amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA gene sequencing. The ARDRA results suggest that the microbial communities on membrane surfaces could be very different from the ones in the suspended biomass. Phylogenetic analysis based on the 16S rRNA gene sequences provided a list of bacteria that might be the pioneers of surface colonization on microfiltration membranes. The results further suggested that research on the mechanisms of cell attachment in such an engineering environment could be critical for future development of appropriate biofouling control strategies.

Epsilon-Proteobacteria is increasingly recognized as an ecologically significant group of bacteria, particularly in deep-sea hydrothermal environments. In this study, we studied the spatial distribution, diversity and physiological characteristics of the epsilon-Proteobacteria in various microbial habitats in the vicinity of a deep-sea hydrothermal vent occurring in the Iheya North field in the Mid-Okinawa Trough, by using culture-dependent and -independent approaches. The habitats studied were inside and outside hydrothermal plume, and annelid polychaete tubes. In addition, we deployed colonization devices near the vent emission. The polychaete tubes harboured physiologically and phylogenetically diverse microbial community. The in situ samplers were predominantly colonized by epsilon-Proteobacteria. Energy metabolism of epsilon-Proteobacteria isolates was highly versatile. Tree topology generated from the metabolic traits was significantly different (P = 0.000) from that of 16S rRNA tree, indicating current 16S rRNA gene-based analyses do not provide sufficient information to infer the physiological characteristics of epsilon-Proteobacteria. Nevertheless, culturability of epsilon-Proteobacteria in various microbial habitats differed among the phylogenetic subgroups. Members of Sulfurimonas were characterized by the robust culturability, and the other phylogenetic subgroups appeared to lose culturability in seawater, probably because of the sensitivity to oxygen. These results provide new insight into the ecophysiological characteristics of the deep-sea hydrothermal vent epsilon-Proteobacteria, which has never been assessed by comparative analysis of the 16S rRNA genes.

We employed a cultivation-independent approach to examine archaeal diversity along a transient soil salinity gradient at Salt Spring in British Columbia, Canada that is routinely eroded due to heavy, recurrent rainfall. Archaeal 16S rRNA gene libraries were created using DNA extracted from three soil samples collected along this gradient. Statistical comparisons indicated similar archaeal richness across sites but, a significant shift in archaeal community composition along the salinity gradient. Seven distinct phylogenetic groups were represented in soil libraries. Haloarchaea were the most commonly sampled group. Other 16S rRNA sequences were related to uncultured Euryarchaeota and Crenarchaeota or halophilic methanogens. Haloarchaeal diversity was remarkably high in soil of elevated salinity compared with previously characterized haloarchaeal communities. Salt Spring haloarchaea were not closely related to known low-salt adapted/tolerant species, suggesting they may be frequently faced with local mortality as a result of frequent declines in soil salinity. We speculate that ecosystem disturbance -- in the form of salinity fluctuations -- is one mechanism for maintaining a diverse community of haloarchaea at Salt Spring.

Mires forming an ecohydrological gradient from nutrient-rich, groundwater-fed mesotrophic and oligotrophic fens to a nutrient-poor ombrotrophic bog were studied by comparing potential methane (CH(4)) production and methanogenic microbial communities. Methane production was measured from different depths of anoxic peat and methanogen communities were detected by detailed restriction fragment length polymorphism (RFLP) analysis of clone libraries, sequencing and phylogenetic analysis. Potential CH(4) production changed along the ecohydrological gradient with the fens displaying much higher production than the ombrotrophic bog. Methanogen diversity also decreased along the gradient. The two fens had very similar diversity of methanogenic methyl-coenzyme M reductase gene (mcrA), but in the upper layer of the bog the methanogen diversity was strikingly lower, and only one type of mcrA sequence was retrieved. It was related to the Fen cluster, a group of novel methanogenic sequences found earlier in Finnish mires. Bacterial 16S rDNA sequences from the fens fell into at least nine phyla, but only four phyla were retrieved from the bog. The most common bacterial groups were Deltaproteobacteria, Verrucomicrobia and Acidobacteria.

Substantial ambiguity exists regarding the phylogenetic status of facultatively psychrophilic luminous bacteria identified as Photobacterium phosphoreum, a species thought to be widely distributed in the world's oceans and believed to be the specific bioluminescent light-organ symbiont of several deep-sea fishes. Members of the P. phosphoreum species group include luminous and non-luminous strains identified phenotypically from a variety of different habitats as well as phylogenetically defined lineages that appear to be evolutionarily distinct. To resolve this ambiguity and to begin developing a meaningful knowledge of the geographic distributions, habitats and symbiotic relationships of bacteria in the P. phosphoreum species group, we carried out a multilocus, fine-scale phylogenetic analysis based on sequences of the 16S rRNA, gyrB and luxABFE genes of many newly isolated luminous strains from symbiotic and saprophytic habitats, together with previously isolated luminous and non-luminous strains identified as P. phosphoreum from these and other habitats. Parsimony analysis unambiguously resolved three evolutionarily distinct clades, phosphoreum, iliopiscarium and kishitanii. The tight phylogenetic clustering within these clades and the distinct separation between them indicates they are different species, P. phosphoreum, Photobacterium iliopiscarium and the newly recognized 'Photobacterium kishitanii'. Previously reported non-luminous strains, which had been identified phenotypically as P. phosphoreum, resolved unambiguously as P. iliopiscarium, and all examined deep-sea fishes (specimens of families Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae and Acropomatidae) were found to harbour 'P. kishitanii', not P. phosphoreum, in their light organs. This resolution revealed also that 'P. kishitanii' is cosmopolitan in its geographic distribution. Furthermore, the lack of phylogenetic variation within 'P. kishitanii' indicates that this facultatively symbiotic bacterium is not cospeciating with its phylogenetically divergent host fishes. The results of this fine-scale phylogenetic analysis support the emerging view that bacterial species names should designate singular historical entities, i.e. discrete lineages diagnosed by a significant divergence of shared derived nucleotide characters.

Diverse methylotrophic bacteria were isolated from the tongue, and supra- and subgingival plaque in the mouths of volunteers and patients with periodontitis. One-carbon compounds such as dimethylsulfide in the mouth are likely to be used as growth substrates for these organisms. Methylotrophic strains of Bacillus, Brevibacterium casei, Hyphomicrobium sulfonivorans, Methylobacterium, Micrococcus luteus and Variovorax paradoxus were characterized physiologically and by their 16S rRNA gene sequences. The type strain of B. casei was shown to be methylotrophic. Enzymes of methylotrophic metabolism were characterized in some strains, and activities consistent with growth using known pathways of C1-compound metabolism demonstrated. Genomic DNA from 18 tongue and dental plaque samples from nine volunteers was amplified by the polymerase chain reaction using primers for the 16S rRNA gene of Methylobacterium and the mxaF gene of methanol dehydrogenase. MxaF was detected in all nine volunteers, and Methylobacterium was detected in seven. Methylotrophic activity is thus a feature of the oral bacterial community.

There is an increasing realization that progress in bacterial ecology can be further advanced by applying theories and models developed in ecology. Consequently, there is a significant need to assess the applicability of such tools, developed specifically for macroorganisms, for investigating the underlying issues that determine bacterial diversity and community assemblage. In this study, we employed the island biogeography species-area model, originally conceived to assess colonization of islands by macroorganisms, to assess bacterial communities colonizing metal-cutting fluids from machines of increasing sump tank size, taking these to be analogous to islands of variable size. This system was selected because it is well studied and compared with other natural bacterial communities has a relatively low (manageable) diversity. Our findings show that island biogeography theory holds for the bacterial communities studied, in that smaller sump tanks contained lower and putatively less stable diversity, and larger sumps had greater diversity and were temporally stable. It was found that the calculated power law indices (i.e. z-values) were similar for all sample sets, and strikingly, typical of those observed in classical ecology. This was not expected as bacteria have significant distinguishing features such as huge population sizes, rapid asexual reproduction and small body size that facilitate dispersal, and are particularly resistant to extinction.

The activity and abundance of methanotrophic bacteria were measured in an acidic and a neutral forest soil. The soils exhibited high uptake rates (>30 microg CH4 m(-2) h(-1)) of atmospheric CH4 at all measurement times throughout the vegetation period. The abundances of various phylogenetic groups of methanotrophs, including some uncultured putative ones, were measured using real-time polymerase chain reaction assays. Each assay specifically targeted the pmoA gene or mmoX gene of a particular group of methanotrophs, or the amoA gene of ammonia-oxidizing bacteria. As yet uncultured methanotrophs of a group previously named 'forest soil cluster' or 'USC alpha' were numerically dominant in the acidic soil, while cultured but taxonomically uncharacterized methanotrophs of a group 'Cluster I' were dominant in the neutral soil. Each group was detected in numbers equivalent to about 10(6) pmoA gene copies per gram dry weight of soil and comprised >90% of the detectable methanotrophic bacteria in the respective soil. As the numbers of ammonia-oxidizing bacteria were similar but not higher, they could not have accounted for the observed CH4 uptake rates due to their low cell-specific CH4 oxidation activity. Based on CH4 flux and bacterial abundance data, estimated cell-specific CH4 oxidation rates of the detected methanotrophic bacteria were 540-800 x 10(-18) mol cell(-1) h(-1), which is high compared with literature values of cultured methanotrophic bacteria. These estimated cell-specific CH4 oxidation rates are sufficiently high to allow not only maintenance but even growth on atmospheric CH4 alone. Transcripts of mRNA of the pmoA gene were detected in the acidic soil, demonstrating that USC alpha methanotrophs expressed pmoA under ambient methane mixing ratios. On the other hand, pmoA transcripts of Cluster I or of other methanotrophic groups were not detectable. Our study suggests that USC alpha and Cluster I methanotrophs are adapted to the low concentration of methane in forest soils by possessing high cell-specific CH4 oxidation activities.

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by <5% of the total OTUs. A comprehensive set of 15 16S and 23S rRNA-targeted oligonucleotide hybridization probes was used to quantify these major groups by dot blot hybridization within 12 digester samples. In contrast to the clone library, Firmicutes and Actinobacteria together accounted for 21.8 +/- 14.9% representing the most abundant phyla. They were surprisingly followed by the Chloroflexi representing 20.2 +/- 4.6% of the total 16S rRNA. The Proteobacteria and the Bacteroidetes group accounted for 14.4 +/- 4.9% and 14.5 +/- 4.3%, respectively, WWE1, a novel lineage, accounted for 11.9 +/- 3.1% while Planctomycetes and Synergistes represented <2% each. Using the novel set of probes we extended the coverage of bacterial populations from 52% to 85.3% of the total rRNA within the digester samples.

A dense 1 m thick layer of phototrophic purple sulfur bacteria is present at the pycnocline (17.8 m depth) in the meromictic South Andros Black Hole cave system (Bahamas). Two purple sulfur bacteria present in samples collected from this layer have been identified as belonging to the family Chromatiaceae. One isolate (BH-1), pink coloured, is non-motile, non-gas vacuolated, 2-3 microm in diameter and surrounded by a capsule. The other isolates (BH-2 and BH-2.4), reddish-brown coloured, are small celled (4 microm x 2 microm), motile by means of a single polar flagellum. In both isolates (BH-1 and BH-2), the intracellular photosynthetic membranes are of the vesicular type and bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series are present. Both isolates grow well in the presence of sulfide and carbon dioxide in the light. During photoautotrophic growth sulfur globules are stored intracellularly as intermediate oxidation products. According to the 16S rRNA gene sequence data the isolates belong to the genera Thiocapsa and Allochromatium. However, at the species level a number of inconsistencies exist between the phenotypic and phylogenetic data, highlighting taxonomic problems within these genera. These inconsistencies may have implications for microbiologists studying the ecology of anoxygenic phototrophs. For ecologists studying the functioning of an ecosystem it may not be particularly important to know whether a specific isolate belongs to one species or another. However, if one wants to study the role of different populations within a particular functional group then the species concept is important. This study demonstrates that further work is still required on the taxonomy of purple sulfur bacteria in order that microbial ecologists are able to accurately identify a population/species isolated from hitherto undescribed aquatic ecosystems.

In this study we analysed the relationship between bacterial community structures and geochemistry of groundwater in a sandstone aquifer (SIReN site) impacted mainly by BTEX hydrocarbons (benzene, toluene, ethylbenzene and xylenes), of which benzene is most abundant. The long-term presence of benzene reduced bacterial diversity: in groundwaters contaminated with more than 1.8 x 10(4) microg l(-1) of benzene, bacterial diversity was half of that in clean groundwaters. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA revealed that the community structures were very similar in uncontaminated groundwaters, whereas communities subjected to long-term benzene contamination were different, not only from uncontaminated groundwater communities, but also from each other. Canonical correspondence analysis of the community profiles and the geochemical data showed that this divergence in community structure was not primarily caused by the direct toxic or stressful effects of benzene, but by the environmental changes brought about by benzene metabolism, in particular a decrease in redox potential.

Detailed studies on the relation of structure and function of microbial communities in a sediment depth profile scarcely exist. We determined as functional aspect the vertical distribution of the acetotrophic and hydrogenotrophic CH4 production activity by measuring production rates and stable 13C/12C-isotopic signatures of CH4 in the profundal sediment of Lake Dagow. The structural aspect was determined by the composition of the methanogenic community by quantifying the abundance of different archaeal groups using 'real-time' polymerase chain reaction and analysis of terminal restriction fragment length polymorphism (T-RFLP). Methane production rates in the surface sediment (0-3 cm depth) were higher in August than in May, but strongly decreased with depth (down to 20 cm). The delta13C of the produced CH4 and CO2 indicated an increase in isotopic fractionation with sediment depth. The relative contribution of hydrogenotrophic to total methanogenesis, which was calculated from the isotopic signatures, increased with depth from about 22% to 38%. Total numbers of microorganisms were higher in August than in May, but strongly decreased with depth. The increase of microorganisms from May to August mainly resulted from Bacteria. The Archaea, on the other hand, exhibited a rather constant abundance, but also decreased with depth from about 1 x 10(8) copies of the archaeal 16S rRNA gene per gram of dry sediment at the surface to 4 x 10(7) copies per gram at 15-20 cm depth. T-RFLP analysis combined with phylogenetic analysis of cloned sequences of the archaeal 16S rRNA genes showed that the methanogenic community consisted mainly of Methanomicrobiales and Methanosaetaceae. The relative abundance of Methanosaetaceae decreased with depth, whereas that of Methanomicrobiales slightly increased. Hence, the vertical distribution of the functional characteristics (CH4 production from acetate versus H2/CO2) was reflected in the structure of the community consisting of acetotrophic (Methanosaetaceae) versus hydrogenotrophic (Methanomicrobiales) phenotypes.

The microbial community associated with the reef building coral Pocillopora damicornis located on the Great Barrier Reef was investigated using culture-independent molecular microbial techniques. The microbial communities of three separate coral colonies were assessed using clone library construction alongside restriction fragment length polymorphism and phylogenetic analysis. Diversity was also investigated spatially across six replicate samples within each single coral colony using 16S rDNA and rpoB-DGGE analysis. Clone libraries demonstrated that the majority of retrieved sequences from coral tissue slurry libraries affiliated with gamma-Proteobacteria. This contrasted with clone libraries of seawater and coral mucus, which were dominated by alpha-Proteobacteria. A number of retrieved clone sequences were conserved between coral colonies; a result consistent with previous studies suggesting a specific microbe-coral association. rpoB-DGGE patterns of replicate tissue slurry samples underestimated microbial diversity, but demonstrated that fingerprints were identical within the same coral. These fingerprints were also conserved across coral colonies. The 16S rDNA-DGGE patterns of replicate tissue slurry samples were more complex, although non-metric multidimensional scaling (nMDS) analysis showed groupings of these banding patterns indicating that some bacterial diversity was uniform within a coral colony. Sequence data retrieved from DGGE analysis support clone library data in that the majority of affiliations were within the gamma-Proteobacteria. Many sequences retrieved also affiliated closely with sequences derived from previous studies of microbial diversity of healthy corals in the Caribbean. Clones showing high 16S rDNA sequence identity to both Vibrio shiloi and Vibrio coralliilyticus were retrieved, suggesting that these may be opportunist pathogens. Comparisons of retrieved microbial diversity between two different sampling methods, a syringe extracted coral mucus sample and an airbrushed coral tissue slurry sample were also investigated. Non-metric multidimensional scaling of clone library data highlighted that clone diversity retrieved from a coral mucus library more closely reflected the diversity of surrounding seawater than a corresponding coral tissue clone library.

Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >or= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.

Among human faecal bacteria, many members of the Clostridium leptum subgroup are fibrolytic and butyrate producing microorganisms thereby contributing to processes important to colonic health. Yet this phylogenetic subgroup remains poorly described to date. To improve detection and description of members of the C. leptum subgroup, the Clep 866 group probe was developed. Its association with probes targeting the Clostridium viride cluster (Cvir 1414) and Eubacterium desmolans species (Edes 635) allowed for the first time the detection of all members found in this phylogenetic group in human faecal microbiota. A species-specific probe was also designed to detect members of the Ruminococcus callidus species (Rcal 733). The design of signature regions was based on alignment of 16S rRNA sequences isolated from faeces of five healthy adults. Furthermore, an oligonucleotide competitor strategy was developed in order to improve the specificity of the probes formerly validated or designed in this study. The oligonucleotide probes were tested using a collection of target and non-target strains using FISH combined with flow cytometry. These new probes were added to a panel of 18 phylogenetic probes selected to describe faecal microbiota composition in 21 human faeces of healthy adults. Clostridium leptum subgroup represented 22% of the total faecal bacteria and codominated with members of Clostridium coccoides group. The cluster Faecalibacterium prausnitzii was the dominant component of the C. leptum subgroup and 20% of the latter subgroup remained unidentified at the species level.

Thirty-three strains classified as Corallococcus coralloides isolated from mostly soil samples in 14 countries of four continents, were subjected to phylogenetic analyses. Based on 16S rDNA analyses the strains form a highly related cluster, sharing above 98.7% sequence similarity. Four groups were recognized within this cluster, only one of which, containing two strains from St. Lucia, Lower Antilles, was exclusively defined by strains from the same sample. The other groups contained members from different countries, even continents. The largest group embraced the type strains of C. coralloides DSM 2259(T) and Corallococcus exiguus 14696(T) which were almost indistinguishable in their 16S rRNA gene sequence. Corallococcus macrosporus DSM 14697(T) grouped outside the C. coralloides cluster, showing a higher relationship to a member of Myxococcus. The topology of the tree generated on the basis of the partial gyrase B (gyrB) gene sequence supports the rRNA gene tree, though some differences in the order of branching were observed. As judged by the binary similarity values the higher resolution power of gyrB sequences was confirmed. From a taxonomic standpoint, the size of myxospores is not a valuable taxonomic criterion, as small- and medium-sized myxospores are members of the same group. If the species status of C. coralloides and C. exiguus is verified by other methods (e.g. DNA-DNA hybridisation, RiboTyping), the genus Corallococcus may embrace a broad range of yet-to-be described novel species. The presence of strains within the same sample displaying higher relatedness to strains from other locations points towards an intensive dispersal of myxospores across continents.

Differences in microenvironment and interactions of microorganisms within and across habitat boundaries should influence structure and diversity of the microbial communities within an ecosystem. We tested this hypothesis using the well characterized gut tract of the European subterranean termite Reticulitermes santonensis as a model. By cloning and sequencing analysis and molecular fingerprinting (terminal restriction fragment length polymorphism), we characterized the bacterial microbiota in the major intestinal habitats - the midgut, the wall of the hindgut paunch, the hindgut fluid and the intestinal protozoa. The bacterial community was very diverse (> 200 ribotypes) and comprised representatives of several phyla, including Firmicutes (mainly clostridia, streptococci and Mycoplasmatales-related clones), Bacteroidetes, Spirochaetes and a number of Proteobacteria, all of which were unevenly distributed among the four habitats. The largest group of clones fell into the so-called Termite group 1 (TG-1) phylum, which has no cultivated representatives. The majority of the TG-1 clones were associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most closely related termites. Overall, differences in diversity between the different gut habitats and the uneven distribution of individual phylotypes support conclusively that niche heterogeneity is a strong determinant of the structure and spatial organization of the microbial community in the termite gut.

A so far uncultured member of the Euryarchaeota was enriched from an anoxic riparian soil and phenotypically characterized using quantitative polymerase chain reaction (qPCR; "real-time PCR"). The microorganism is related to the Thermoplasmatales and belongs to Rice Cluster III (RC-III). Enrichment cultures utilized yeast extract (YE) by transiently accumulating acetate as major fermentation product, which was subsequently converted to methane. The abundance of RC-III archaea within the enrichment cultures was quantified by analysis of the terminal restriction fragment length polymorphism (T-RFLP) and by qPCR. We developed qPCR assays targeting the 16S rRNA genes (16S rDNA) specific for RC-III as well as for the Archaea in general. The enrichment cultures consisted of a mixed methanogenic community of Bacteria and Archaea, the latter consisting of up to 60% of members of RC-III. The other archaea belonged to Methanosarcinaceae, Methanomicrobiaceae and Methanobacteriaceae. The enriched RC-III archaea were represented by two sequences (LL25A, LL37A) that were highly similar to each other and to those detected in the soil inoculum (>98% similarity). However, the 16S rDNA copy numbers of RC-III archaea were about 1000-fold lower than those of Bacteria. Nevertheless, we were able to estimate growth parameters and physiological properties of one of the enriched RC-III archaea (LL25A) by measuring the increase of 16S rDNA copy numbers specific for this group under different growth conditions. The enriched RC-III archaeon grew optimally at temperatures between 20 and 30 degrees C and neutral pH using YE, meat extract, peptone or tryptone under anoxic conditions. Doubling time was approximately 3 days. No proliferation was detected on carbohydrates, amino acids, fatty acids, glycerol, alcohols, aromatic compounds, purine and pyrimidine bases or pyruvate. Various exogenous electron acceptors (e.g. ferric iron, S(0)) did not support growth on YE. Proliferation of the enriched RC-III archaeon was hardly affected by the antibiotics ampicillin, kanamycin and streptomycin. These findings suggest that the enriched archaeon is a mesophilic anaerobe, which can grow heterotrophically on peptides. Further enrichment on peptone and kanamycin eventually allowed the microscopic detection of coccoid cells stained by fluorescence in situ hybridization (FISH).

We investigated the influence of desiccation frequency, indicated by tidal position, on microbial community structure, diversity and richness of microbial mats. We independently characterized cyanobacterial, bacterial and archaeal communities, and their spatial variability for two distinct microbial mat systems: subtidal hypersaline mats and intertidal sand flat mats. Community fingerprints based on 16S rDNA were obtained via denaturing gradient gel electrophoresis using polymerase chain reaction primers specific for each group. Fingerprints for all three groups were consistently similar [> or =85% according to Weighted Pair Group with Arithmetic Mean (WPGMA) analysis] along a 1-km-long transect in subtidal mats. Here, pair-wise comparison analysis yielded minimal variation in diversity and richness for all groups. Fingerprints of three sites along an intertidal transect were heterogenous (> or =32% similarity according to WPGMA analysis) with clear shifts in community structure in all three microbial groups. Here, all groups exhibited statistically significant decreases in richness and diversity with tidal height (as desiccation frequency increases). Regression analysis yielded a strong correlation between diversity or richness estimates and position along the tidal gradient, for both Archaea and Bacteria, with Cyanobacteria exhibiting a weaker correlation. These results suggest that desiccation frequency can shape the structure of microbial mat communities, with Archea being least tolerant and Cyanobacteria most tolerant.

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.

The development of soil archaeal community structures in relation to primary succession in bulk and rhizosphere soil was examined across the forefield of the receding Rotmoosferner glacier in Austria. Using cloning and denaturing gradient gel electrophoresis (DGGE) analysis of reverse transcription polymerase chain reaction (RT-PCR) products of extracted 16S rRNA, archaeal community structure was compared over a chronosequence representing approximately 150 years of soil development and to reference sites outside the glacier forefield, representing soil exposed for approximately 9500 years. Archaeal community composition was found to be dominated by members of the non-thermophilic or Group 1 Crenarchaeota, where a dramatic yet highly structured successional sequence was observed. Succession over the 150 years sequence could be identified as occurring in three stages, each of which had a phylogenetically distinct 1.1b crenarchaea community with those organisms present in pioneering and intermediate stages belonging to a lineage distinct from those in developed soils. Climax communities also contained organisms belonging to three other major non-thermophilic crenarchaeal lineages. Comparison of archaeal communities in the rhizosphere indicated that plant species composition was not the major driver of specific crenarchaeal populations. These results indicate the potential role of soil crenarchaea in the development of soil substrates, as well as ecological diversity within and between major Group 1 lineages.

We have studied the diversity of pelagic cyanobacteria in Lake Loosdrecht, The Netherlands, through recovery and analysis of small subunit ribosomal RNA gene sequences from lake samples and cyanobacterial isolates. We used an adapted protocol for specific amplification of cyanobacterial rDNA for denaturing gradient gel electrophoresis (DGGE) analysis. This protocol enabled direct comparison of cyanobacterial community profiles with overall bacterial profiles. The theoretical amplification specificity of the primers was supported by sequence analysis of DNA from excised DGGE bands. Sequences recovered from these bands, in addition to sequences obtained by polymerase chain reaction (PCR) and cloning from lake DNA as well as from cyanobacterial isolates from the lake, revealed a diverse consortium of cyanobacteria, among which are representatives of the genera Aphanizomenon, Planktothrix, Microcystis and Synechococcus. One numerically important and persistent cyanobacterium in the lake, Prochlorothrix hollandica, appeared to co-occur with an unknown but related species. However, the lake is dominated by filamentous species that originally have been termed 'Oscillatoria limnetica-like'. We show that this is a group of several related cyanobacteria, co-occurring in the lake, which belong to the Limnothrix/Pseudanabaena group. The available variation among the coexisting strains of this group can explain the persistent dominance of the group under severe viral pressure.

The human-specific HF183 Bacteriodes 16S rRNA genetic marker can be used to detect human faecal pollution in water environments. However, there is currently no method to quantify the prevalence of this marker in environmental samples. We developed a real-time polymerase chain reaction (PCR) assay using SYBR Green I detection to quantify this marker in faecal and environmental samples. To decrease the amplicon length to a suitable size for real-time PCR detection, a new reverse primer was designed and validated on human and animal faecal samples. The use of the newly developed reverse primer in combination with the human-specific HF183 primer did not decrease the specificity of the real-time PCR assay but a melting curve analysis must always be included. This new assay was more sensitive than conventional PCR and highly reproducible with a coefficient of variation of less than 1% within an assay and 3% between assays. As the Bacteroides species that carries this human-specific marker has never been isolated, a bacteria real-time assay was used to determine the detection efficiency. The estimated detection efficiency in freshwater ranged from 78% to 91% of the true value with an average detection efficiency of 83+/-4% of the true value. Using a simple filtration method, the limit of quantification was 4.7+/-0.3x10(5) human-specific Bacteroides markers per litre of freshwater. The aerobic incubation of the human-specific Bacteroides marker in freshwater for up to 24 days at 4 and 12 degrees C, and up to 8 days at 28 degrees C, indicated that the marker persisted up to the end of the incubation period for all incubation temperatures.

The 'Synergistes' group, which apparently represents an as yet unnamed division of the bacteria, was explored in 93 anaerobic environments (guts, soils, digestors, etc.). From 16S rDNA gene-targeted polymerase chain reaction (PCR) assays, this group appeared to be present in 90% of the anaerobic microbial ecosystems analysed. The phylogeny of 103 16S rDNA sequences from 30 ecosystems showed a strong link between 16S rDNA sequences and given ecosystems. 'Synergistes' 16S rDNA sequences from animal sources (termites, guinea pigs, pigs, birds, etc.) formed clustered phylogenetical groups. 'Synergistes' groups were also associated either with anaerobic digestors and soils or with thermophilic conditions. Sequences available from the DNA database were consistent with the results. These results show the wide diversity of the 'Synergistes' division as well as the specific ecological niche of each 16S rDNA sequences.

The salt composition of 14 acidic lakes was analysed, and their microbial communities were compared by their 16S rDNA-based single-strand conformation polymorphism (SSCP) fingerprints. The lakes were grouped into three chemically distinct types, and operational taxonomic units (OTUs) were identified in the 16S rDNA SSCP fingerprints. Discriminant analysis between these groups using a multivariate approach showed that not a single organism, but rather a set of seven OTUs (Ferromicrobium acidophilum, Bacillus pumilus, Acidophilium organovorum, Paenibacillus chibensis, Acidocella sp. and two different alpha-Proteobacteria), was found to be indicative for a given lake type. We conclude that, for these lakes, not single indicator strains but indicator groups exist that are controlled by the ionic composition of the lakes.

The distribution of bacterial communities terminal restriction fragment length polymorphism (T-RFLP) fingerprint patterns was evaluated at three proximal hydrocarbon-contaminated sites located within the harbour of Messina. In order to analyse the short-term variability of the individual terminal restriction fragment (T-RF) patterns, water samples were collected at the three sites on three occasions within 3 months (T(0), T(90) and T(91)). Four sample sizes, from 50 to 1000 ml for each collected sample, were analysed separately (36 total analysed samples) to evaluate the relationship between the sample size and the bacterial diversity estimates. The dominant T-RF groups mostly belonged to signatures of putative hydrocarbon-degrading bacteria, as revealed by the virtual analysis of the obtained bands. In order to test whether significant differences were occurring between the analysed samples, the Kruskal-Wallis non-parametric test was applied to the T-RF data set. Neither significant influence of the sample size nor short spatial variability within the three sampled sites was detected for each sampling time. On the contrary, significant temporal changes in the diversity of the bacterial communities were observed. These results were confirmed by the non-metric multidimensional scales (nMDS) analysis of the whole set of samples, which indicated three main groups corresponding to the three different sampling times. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring polluted marine environments, typically characterized by low diversity and high relative abundances of a few dominant groups.

Many Archaea, in contrast to bacteria, produce a high proportion of leaderless transcripts, show a wide variation in their consensus Shine-Dalgarno (S-D) sequences and frequently use GUG and UUG start codons. In order to understand the basis for these differences, 18 complete archaeal genomes were examined for sequence signals that are positionally conserved upstream from genes. These functional motifs include box A promoter sequences for leaderless transcripts and S-D sequences for transcripts with leaders. Most of the box A sequences were preceded by a BRE-like motif and followed by a previously undetected A/T peak centred on position -10. Moreover, the sequence of the predominant S-D motifs in an archaeon is shown to depend on the precise number of nucleotides between the conserved anti-S-D CCUCC sequence and the 3'-terminal nucleotide of 16S RNA. Correlations with phylogenetic trees, constructed for the 18 Archaea, reveal that usage of high levels of both S-D motifs, and GUG and UUG start codons occurs exclusively in the shorter branched Archaea. High levels of leaderless transcripts are found in the longer branched Archaea.

Three novel strains of methylotrophic Afipia felis were isolated from several locations on Signy Island, Antarctica, and a fourth from estuary sediment from the River Douro, Portugal. They were identified as strains of the alpha-2 proteobacterium A. felis by 16S rRNA gene sequence analysis. Two strains tested were shown to contain the fdxA gene, diagnostic for A. felis. All strains grew with methanesulfonate (and two strains with dimethylsulfone) as sole carbon substrate. Growth on methanesulfonate required methanesulfonate monooxygenase (MSAMO), using NADH as the reductant and stimulated by reduced flavin nucleotides and Fe(II). Polymerase chain reaction amplification of DNA from an Antarctic strain showed a typical msmA gene for the alpha-hydroxylase of MSAMO, and both Antarctic and Portuguese strains contained mxaF, the methanol dehydrogenase large subunit gene. This is the first report of methanesulfonate-degrading bacteria from the Antarctic and of methylotrophy in Afipia, and the first description of any bacterium able to use both methanesulfonate and dimethylsulfone. In contrast, the type strain of A. felis DSM 7326(T) was not methylotrophic, but grew in defined mineral medium with a wide range of single simple organic substrates. Free-living Afipia strains occurring widely in the natural environment may be significant as methylotrophs, degrading C(1)-sulfur compounds, including the recalcitrant organosulfur compound methanesulfonate.

The capability of planar rRNA-based oligonucleotide microarrays for single-base-pair discrimination was evaluated using an approach that compares the non-equilibrium dissociation profiles and dissociation temperatures (Tds) of all probe-target duplexes simultaneously. Three sets of 16S rRNA gene specific probes at different levels of specificity were used along with their counter probes for individual sets having either one or two mismatches (MM) to their targets at specific external (next to terminus) and various internal positions. Criteria based on the Td approach and a discrimination index (DI) were proven to be competent in discriminating PM from internal MM duplexes, but not always for external MM duplexes. Maximal DI for separating PM duplexes from ones with two and one internal MM usually occurred at temperatures approximately 5-10 degrees C and 10-15 degrees C, respectively, higher than the Tds of the PM duplexes. Washing buffer type and salt concentration, and MM number and position were shown statistically to affect dissociation profiles, Td, and single-base-pair discriminating capability. The reusability potential of the planar microchip was further demonstrated.

Marine seeps introduce significant amounts of hydrocarbons into oceans and create unusual habitats for microfauna and -flora. In the vicinity of chronic seeps, microbes likely exert control on carbon quality entering the marine food chain and, in turn, hydrocarbons could influence microbial community composition and diversity. To determine the effects of seep oil on marine sediment bacterial communities, we collected sediment piston cores within an active marine hydrocarbon seep zone in the Coal Oil Point Seep Field, at a depth of 22 m in the Santa Barbara Channel, California. Cores were taken adjacent to an active seep vent in a hydrocarbon volcano, on the edge of the volcano, and at the periphery of the area of active seepage. Bacterial community profiles were determined by terminal restriction fragment length polymorphisms (TRFLPs) of 16S ribosomal genes that were polymerase chain reaction (PCR)-amplified with eubacterial primers. Sediment carbon content and C/N ratio increased with oil content. Terminal restriction fragment length polymorphisms suggested that bacterial communities varied both with depth into sediments and with oil concentration. Whereas the apparent abundance of several peaks correlated positively with hydrocarbon content, overall bacterial diversity and richness decreased with increasing sediment hydrocarbon content. Sequence analysis of a clone library generated from sediments collected at the periphery of the seep suggested that oil-sensitive species belong to the gamma Proteobacteria and Holophaga groups. These sequences were closely related to sequences previously recovered from uncontaminated marine sediments. Our results suggest that seep hydrocarbons exert a strong selective pressure on bacterial communities in marine sediments. This selective pressure could, in turn, control the effects of oil on other biota in the vicinity of marine hydrocarbon seeps.

An extensive microcosm survey of perchlorate-contaminated sites was undertaken to assess the ability of indigenous microorganisms to degrade perchlorate. Samples from 12 contaminated sites and from one pristine location were analysed. Perchlorate was degraded to below detection limit in all electron donor-amended microcosms. Perchlorate-reducing microorganisms (PRMs) were numerous at most of these sites. Sixteen distinct PRMs were isolated that were phylogenetically related to either Dechloromonas in the Beta Proteobacteria (9/16 isolates) or to Azospirillum in the Alpha Proteobacteria (7/16 isolates). The majority of previously isolated PRMs are in the Beta Proteobacteria related to Dechloromonas or Dechlorosoma. This study indicates that PRMs of the genus Azospirillum may be more prevalent at contaminated sites than the current record of isolates suggests. Cell yields, electron donor to perchlorate ratios and maximum specific growth rates were similar among the isolates and similar to the few previously published values. However, the Monod half-saturation constants for perchlorate for the two Azospirillum isolates characterized were lower than those measured for other genera, suggesting that they may be more effective at low concentrations of perchlorate. These results extend the current understanding of PRMs from diverse environments and provide added confidence that microbial perchlorate reduction is ubiquitous, even at highly contaminated sites, and can be harnessed effectively for bioremediation.

In a contaminated water-table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i). communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii). after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii). after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity (H) and the evenness index (E), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate-reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.

Anoxic soils in river floodplains (or riparian soils) are a source of methane emission. However, little is known about the ecology and community structure of archaeal methanogenic microbes, which are a crucial component of methane flux in those habitats. We studied the archaeal community in the vertical profile of four different sites along the River Waal in the Netherlands. These sites differ in their annual flooding regime ranging from never or seldom to permanently flooded. The archaeal community structure has been characterized by terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of the archaeal SSU rRNA gene and the mcrA gene. The latter gene codes for the alpha-subunit of methyl-coenzyme M reductase. Additionally, the potential methanogenic activity was determined by incubation of soil slurries under anoxic conditions. The community composition differed only slightly with the depth of the soil (0-20 cm). However, the diversity of archaeal SSU rRNA genes increased with the frequency of flooding. Terminal restriction fragment length polymorphism analysis of mcrA gene amplicons confirmed the results concerning methanogenic archaea. In the never and rarely flooded soils, crenarchaeotal sequences were the dominant group. In the frequently and permanently flooded soils, Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae and the uncultured Rice Clusters IV and VI (Crenarchaeota) were detectable independently from duration of anoxic conditions. Methanosaetaceae, on the other hand, were only found in the permanently and frequently flooded soils under conditions where concentrations of acetate were < 30 microM. The results indicate that methanogens as well as other archaea occupy characteristic niches according to the flooding conditions in the field. Methanosaetaceae, in particular, seem to be adapted (or proliferate at) to low acetate concentrations.

Molecular microbial ecology has revealed remarkable biodiversity - prokaryotic and eukaryotic - in numerous soil environments. However, no culture-independent surveys of the termitosphere exists, although termites dominate tropical rainforests. Here, we focused on soil feeders, building nests with their soil-born faeces, enriched with clay-organic complexes, thus contributing to the improvement of soil fertility. In order to assess the fungal community composition of these termitaries compared with soils not foraged by termites, samples of the two types were collected in the Lopé rainforest, Gabon, and processed for generation of fungal internal transcribed spacer (ITS) clone libraries. Although primers were universal, most of the recovered sequences represented Ascomycete that were previously uncharacterized and the proportions of which reached 72.5% in soils and 80% in termitaries. Their affiliation with identified fungi was analysed in performing a phylogenetic tree based on 5.8S rDNA. Furthermore, the ascomycete communities of soil-feeding termitaries and soils shared only 6.3% of sequences. This discrepancy of composition between soil and nest may result from the building behaviour of termites, as the organic matter in the nest is chemically modified, and some vacant ecological microniches are available for more specialized fungi.

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.