Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and cultivation

Fecal microbial diversity in a strictly vegetarian woman was determined by the 16S rDNA library method, terminal restriction fragment length polymorphism (T-RFLP) analysis and a culture-based method. The 16S rDNA library was generated from extracted fecal DNA, using bacteria-specific primers. Randomly selected clones were partially sequenced. T-RFLP analysis was performed using amplified 16S rDNA. The lengths of T-RF were analyzed after digestion by HhaI and MspI. The cultivated bacterial isolates were used for partial sequencing of 16S rDNA. Among 183 clones obtained, approximately 29% of the clones belonged to 13 known species. About 71% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). A total of 55 species or phylotypes were identified among the 16S rDNA library, while the cultivated isolates included 22 species or phylotypes. In addition, many new phylotypes were detected from the 16S rDNA library. The 16S rDNA library and isolates commonly included the Bacteroides group, Bifidobacterium group, and Clostridium rRNA clusters IV, XIVa, XVI and XVIII. T-RFLP analysis revealed the major composition of the vegetarian gut microbiota were Clostridium rRNA subcluster XIVa and Clostridium rRNA cluster XVIII. The dominant feature of this strictly vegetarian gut microbiota was the detection of many Clostridium rRNA subcluster XIVa and C. ramosum (Clostridium rRNA cluster XVIII).     

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.     

Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods

The human gut microbiota from three healthy subjects were compared by the use of a sequence analysis of 16S rDNA libraries and a culture-based method. Direct counts ranged from 1.9 X 10" to 4.0 X 10" cells/g (wet weight), and plate counts totaled 6.6 X 10(10) to 1.2 X 10(11) CFU/g (wet weight). Sixty to seventy percent of the bacteria in the human intestinal tract cannot be cultured with currently available methods. The 16S rDNA libraries from three subjects were generated from total community DNA in the intestinal tract with universal primer sets. Randomly selected clones were partially sequenced. All purified colonies detected from the surface of the agar plate were used for a partial sequencing of 16S rDNA. On the basis of sequence similarities, the clones and colonies were classified into several clusters corresponding to the major phylum of the domain Bacteria. Among a total of 744 clones obtained, approximately 25% of them belonged to 31 known species. About 75% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). The predominant intestinal microbial community consisted of 130 species or phylotypes according to the sequence data in this study. The 16S rDNA libraries and colonies included the Bacteroides group, Streptococcus group, Bifidobacterium group, and Clostridium rRNA clusters IV, IX, XIVa, and XVIII. Moreover, several previously uncharacterized and uncultured microorganisms were recognized in clone libraries and colonies. Our results also showed marked individual differences in the composition of intestinal microbiota.     

The relationship between microbiota and polyamine concentration in the human intestine: a pilot study

The fecal microbiota of 10 hospitalized elderly subjects and 14 healthy adults were analyzed by terminal-restriction fragment length polymorphism (T-RFLP) analysis using Hha I, Msp I, Hae III, and Alu I, as well as fecal polyamine (PA) concentration. The T-RFLP profiles of the fecal microbiota of the subjects were roughly divided into 2 clusters-I (9 out of 11 were derived from hospitalized elderly subjects) and II (12 out of 13 were derived from healthy adults). The average concentration of putrescine in Cluster II was 5.8 times higher than that of putrescine in Cluster I (P=0.0015). Using a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota, the terminal-restriction fragments (T-RFs) characteristically detected in the case of subjects with high fecal PA concentration were predicted to be derived from bacterial species and phylotypes belonging to Clostridium subcluster XIVa, particularly including Clostridium xylanolyticum, Clostridium saccharolyticum, the uncultured human intestinal bacterium clone JW1H4 (a relative of Desulfotomaculum guttoideum), Roseburia intestinalis, the uncultured bacterium clone 41F10 (a relative of Eubacterium ramulus), Roseburia cecicola, Ruminococcus obeum and its relatives. From these results, we concluded that fecal microbiota may be linked with fecal PA concentration and that some bacterial species belonging to Clostridium subcluster XIVa may play a major role in the control of intestinal PA concentration in humans.     

Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota

Various molecular-biological approaches using the 16S rRNA gene sequence have been used for the analysis of human colonic microbiota. Terminal- restriction fragment length polymorphism (T-RFLP) analysis is suitable for a rapid comparison of complex bacterial communities. Terminal-restriction fragment (T-RF) length can be calculated from a known sequence, thus one can predict bacterial species on the basis of their T-RF length by this analysis. The aim of this study was to build a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM), and to demonstrate the effectiveness of PAD-HCM compared with the results of 16S rRNA gene clone library analysis. PAD-HCM was completed to include 342 sequence data obtained using four restriction enzymes. Approximately 80% of the total clones detected by 16S rRNA gene clone library analysis were the same bacterial species or phylotypes as those assigned from T-RF using PAD-HCM. Moreover, large T-RFs consisted of common species or phylotypes detected by both analytical methods. All pseudo-T-RFs identified by mung bean nuclease digestion could not be assigned to a bacterial species or phylotype, and this finding shows that pseudo-T-RFs can also be predicted using PAD-HCM. We conclude that PAD-HCM built in this study enables the prediction of T-RFs at the species level including difficult-to-culture bacteria, and that it is very useful for the T-RFLP analysis of human colonic microbiota.     

Phylogenetic analysis of partial bacterial 16S rDNA sequences of tropical grass pasture soil under Acacia tortilis subsp. raddiana in Senegal

We used direct recovery of bacterial 16S rRNA gene sequences to investigate the bacterial diversity under Acacia tortilis subsp. raddiana, a legume tree naturally growing in the dry land part of Senegal (West Africa). Microbial DNA was purified directly from soil samples and subjected to PCR with primers specific for bacterial 16S rRNA gene sequences. 16S rDNA clone libraries were constructed from two soil samples taken at two dates, i.e. June 25th 1999 (dry season) and August 28th 1999 (rainy season) at depths of 0.25-0.50 m and at 3 m distance from the stem. The 16S rDNA of 117 clones was partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity (100 phylotypes). Comparative sequence analysis of these clones identified members of the Gammaproteobacteria (35% of the phylotypes) as the most important group, followed by the Firmicutes division with 24%. Alphaproteobacteria, Betaproteobacteria, Acidobacteria and Actinobacteria were found to be less represented. Our data suggest that bacterial communities under Acacia tortilis subsp. raddiana might differ according to the season. The relative compositions of the populations is different in both samples: the Acidobacteria are present in a much higher percentage in the dry season than in the rainy season sample while the inverse effect is observed for the members of the other groups. Within the Gammaproteobacteria we found a shift between the dry season and the rainy season from pseudomonads to Acinetobacter and Escherichia related organisms.     

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.     

Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries

The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries. Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected. The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles. The majority of clones obtained in this study belonged to the Clostridium coccoides (C. coccoides) group, the Bacteroides group or the Lactobacillus group. Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C. coccoides and Bacteroides groups. The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries. T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data. T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.     

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.     

Novel Archaea and Bacteria Dominate Stable Microbial Communities in North America’s Largest Hot Spring

Boiling Springs Lake is an approximately 12,000 m(2), 55 degrees C, pH 2 thermal feature located in Lassen Volcanic National Park in northern California, USA. We assessed the microbial diversity in the lake by analyzing approximately 500 sequences from clone libraries constructed using three different primer sets targeted at 16S rRNA genes and one targeted at 18S rRNA genes. We assessed the stability of the microbial community by constructing terminal restriction fragment length polymorphism (T-RFLP) profiles using DNA extracts collected in four separate years over a 7-year period. The four most prevalent phylotypes in the clone libraries shared an average approximately 85% sequence identity with their closest cultured relatives, and three fourths of the prokaryotic sequences shared less than 91% identity. Phylogenetic analyses revealed novel lineages devoid of cultivated representatives in the Bacterial and Archaeal domains. Many detected phylotypes were related to taxonomically diverse genera previously associated with high-temperature environments, while others were related to diverse Proteobacteria and Firmicutes that would not be expected to grow within BSL conditions. All of the 18S rRNA sequences most closely matched fungi in the phyla Ascomycota and Basidiomycota (91-99% identity). T-RFLP detected fragments corresponding to the most prevalent phylotypes detected in 16S rRNA gene libraries. The T-RFLPs from separate years were similar, and the water-derived T-RFLPs were similar to the sediment-derived (average pairwise Sorenson's similarity index of 0.74, and 0.78, respectively). Collectively, these results indicate that a stable community of diverse novel microorganisms exists in Boiling Springs Lake.     

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.     

Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the beta-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by beta-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.     

Culture-independent phylogenetic analysis of the faecal flora of the rat

The dominant faecal flora of the rat was determined using randomly cloned 16S rDNA comparative sequence analysis. A total of 109 near full-length 16S rDNA clones were sequenced, representing 69 unique 16S rRNA phylotypes or operational taxonomic units (OTUs). Estimates of species richness indicated that approximately 338 species were present in the faeces, suggesting that only 20% of species were identified. Only two of 39 Gram-negative clones aligned with previously cultured species, the remainder fell into a separate lineage within the Bacteroides-Cytophaga phylum. Several clones within this new group were related to 16S rDNA sequences previously identified from mouse faeces. Lactobacilli were the most abundant Gram-positive species, representing 23% of the total clones but only 7% of OTUs. The remaining Gram-positive clones were distributed among the Clostridium coccoides group (9%), the Clostridium leptum subgroup (18%), and throughout the low GC Gram-positive bacteria (13%). The majority of OTUs (63/69 or 91%) were less than 97% homologous to previously cultured bacteria. Faecal samples were also cultured using a variety of anaerobic media. With the exception of the lactobacilli, the cultured isolates demonstrated low species diversity and poorly reflected the population, as defined through comparative sequence analysis.     

Formation of pseudo-terminal restriction fragments,a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.     

A molecular phylogenetic survey of sea-ice microbial communities (SIMCO)

16S rDNA clone library analysis was used to identify bacterial biodiversity in a variety of sea-ice microbial communities (SIMCO). DNA was extracted from seven Antarctic sea-ice samples and one Arctic sea-ice sample and 16S rDNA PCR-amplified using universal and Archaea-specific primers. Recombinant 16S rDNA clones were obtained and dereplicated using restriction fragment length polymorphism analysis (RFLP). After RFLP analysis, 100 distinct phylotypes (a unique clone or group of clones with sequence similarity of >0.98) were defined. From the clone libraries 16S rDNA sequences of bacterial and eukaryotic origin were detected, however Archaea were not detected either with universal or Archaea-specific 16S rDNA primer sets. Bacterial phylotypes grouped within the alpha and gamma proteobacteria, the Cytophaga-Flavobacterium-Bacteroides division, the Gram-Positive bacteria and the orders Chlamydiales and Verrucomicrobiales. The majority of bacterial phylotypes were affiliated with heterotrophic taxa and many grouped closely with cultivated genera and species. Eukaryotic clones were affiliated with a variety of autotrophic and heterotrophic nanoplankton and included a large number of chloroplast 16S rDNA genes. The findings of this investigation corroborated culture data indicating bacterial biodiversity increased in SIMCO displaying high levels of primary production, however the bacterial communities within SIMCO were highly heterogeneous at the genus/species-level between different samples. A comparison of Antarctic and Arctic SIMCO revealed certain sea-ice dwelling bacterial genera are common at both poles.     

T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages

The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.     

Replicability of dominant bacterial populations after long-term surfactant-enrichment in lab-scale activated sludge

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.     

Comparative 16S rDNA and 16S rRNA sequence analysis indicates that Actinobacteria might be a dominant part of the metabolically active bacteria in heavy metal-contaminated bulk and rhizosphere soil

Bacterial diversity in 16S ribosomal DNA and reverse-transcribed 16S rRNA clone libraries originating from the heavy metal-contaminated rhizosphere of the metal-hyperaccumulating plant Thlaspi caerulescens was analysed and compared with that of contaminated bulk soil. Partial sequence analysis of 282 clones revealed that most of the environmental sequences in both soils affiliated with five major phylogenetic groups, the Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Acidobacteria and the Planctomycetales. Only 14.7% of all phylotypes (sequences with similarities> 97%), but 45% of all clones, were common in the rhizosphere and the bulk soil clone libraries. The combined use of rDNA and rRNA libraries indicated which taxa might be metabolically active in this soil. All dominant taxa, with the exception of the Actinobacteria, were relatively less represented in the rRNA libraries compared with the rDNA libraries. Clones belonging to the Verrucomicrobiales, Firmicutes, Cytophaga-Flavobacterium-Bacteroides and OP10 were found only in rDNA clone libraries, indicating that they might not represent active constituents in our samples. The most remarkable result was that sequences belonging to the Actinobacteria dominated both bulk and rhizosphere soil libraries derived from rRNA (50% and 60% of all phylotypes respectively). Seventy per cent of these clone sequences were related to the Rubrobacteria subgroups 2 and 3, thus providing for the first time evidence that this group of bacteria is probably metabolically active in heavy metal-contaminated soil.     

Composition and Diversity of Microbial Communities Recovered from Surrogate Minerals Incubated in an Acidic Uranium-Contaminated Aquifer

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the β-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by β-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.