Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal

Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0-0.25 m and 0.25-0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.     

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.     

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.     

Symbiotic and taxonomic diversity of rhizobia isolated from Acacia tortilis subsp. raddiana in Africa

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana from various sites in the North and South of Sahara was analyzed for their diversity at both taxonomic and symbiotic levels. On the basis of whole cell protein (SDS-PAGE) and 16S rDNA sequence analysis, most of the strains were found to belong to the Sinorhizobium and Mesorhizobium genera where they may represent several different genospecies. Despite their chromosomal diversity, most A. tortilis Mesorhizobium and Sinorhizobium symbionts exhibited very similar symbiotic characters. Nodulation tests showed that the strains belong to the Acacia-Leucaena-Prosopis nodulation group, although mainly forming non-fixing nodules on species other than A. tortilis. Most of the strains tested responded similarly to flavonoid nod gene inducers, as estimated by using heterologous nodA-lacZ fusions. Thin layer chromatography analysis of the Nod factors synthesized by overproducing strains showed that most of the strains exhibited similar profiles. The structures of Nod factors produced by four different Sinorhizobium sp. strains were determined and found to be similar to other Acacia-Prosopis-Leucaena nodulating rhizobia of the Sinorhizobium-Mesorhizobium-Rhizobium branch. They are chitopentamers, N-methylated and N-acylated by common fatty acids at the terminal non reducing sugar. The molecules can also be 6-O sulfated at the reducing end and carbamoylated at the non reducing end. The phylogenetic analysis of available NodA sequences, including new sequences from A. tortilis strains, confirmed the clustering of the NodA sequences of members of the Acacia-Prosopis-Leucaena nodulation group.     

Molecular analysis of fecal microbiota in elderly individuals using 16S rDNA library and T-RFLP

Fecal microbiota in six elderly individuals were characterized by the 16S rDNA libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis. Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets from total genomic DNA extracted from feces of three elderly individuals. These clones were partially sequenced (about 500 bp). T-RFLP analysis was performed using 16S rDNA amplified from six subjects. The lengths of the terminal restriction fragment (T-RF) were analyzed after digestion by HhaI and MspI. Among 240 clones obtained, approximately 46% belonged to 27 known species. About 54% of the other clones were 56 novel "phylotypes" (at least 98% homology of clone sequence). These libraries included 83 species or phylotypes. In addition, about 13% (30 phylotypes) of these phylotypes were newly discovered in these libraries. A large number of species that are not yet known exist in the feces of elderly individuals. 16S rDNA libraries and T-RFLP analysis revealed that the majority of bacteria were Bacteroides and relatives, Clostridium rRNA cluster IV, IX, Clostridium rRNA subcluster XIVa, and "Gammaproteobacteria". The proportion of Clostridium rRNA subcluster XIVa was lower than in healthy adults. In addition, although Ruminococcus obeum and its closely related phylotypes were detected in high frequency in healthy young subjects, hardly any were detected in our elderly individuals. "Gammaproteobacteria" were detected at high frequency.     

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.     

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

北极太平洋扇区海洋沉积物细菌多样性的系统发育分析

对北极太平洋扇区3个不同深度的海洋沉积物样品,采用PCR结合变性梯度凝胶电泳(DGGE)技术进行细菌16S rRNA基因V3区序列的系统发育分析。结果表明,同一个沉积物样品不同层次的DGGE电泳图谱不完全相同。从3个沉积物样品中共获得50条序列,大部分序列与从海洋环境尤其海洋沉积物获得的细菌16S rDNA序列相似性较高(88%~100%),归属于变形细菌(Proteobacteria)的gamma亚群、alpha亚群、beta亚群、epsilon亚群、delta亚群,Cytophaga_Flavobacterium_Bacteroides(CFB)群细菌和高G C含量的革兰氏阳性细菌等系统分类群,其中变形细菌(Proteobacteria)的gamma亚群为沉积物中的优势细菌类群。     

Prokaryotic diversity in continuous cropping and rotational cropping soybean soil

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

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).     

Distinctive gut microbiota of honey bees assessed using deep sampling from individual worker bees

Surveys of 16S rDNA sequences from the honey bee, Apis mellifera, have revealed the presence of eight distinctive bacterial phylotypes in intestinal tracts of adult worker bees. Because previous studies have been limited to relatively few sequences from samples pooled from multiple hosts, the extent of variation in this microbiota among individuals within and between colonies and locations has been unclear. We surveyed the gut microbiota of 40 individual workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Over 99% of these sequences belonged to clusters for which the first blastn hits in GenBank were members of the known bee phylotypes. Four phylotypes, one within Gammaproteobacteria (corresponding to "Candidatus Gilliamella apicola") one within Betaproteobacteria ("Candidatus Snodgrassella alvi"), and two within Lactobacillus, were present in every bee, though their frequencies varied. The same typical bacterial phylotypes were present in all colonies and at both sites. Community profiles differed significantly among colonies and between sites, mostly due to the presence in some Arizona colonies of two species of Enterobacteriaceae not retrieved previously from bees. Analysis of Sanger sequences of rRNA of the Snodgrassella and Gilliamella phylotypes revealed that single bees contain numerous distinct strains of each phylotype. Strains showed some differentiation between localities, especially for the Snodgrassella phylotype.     

Phylogenetic diversity of microorganisms associated with the deep-water sponge Baikalospongia intermedia

The diversity of bacteria associated with deep-water sponge Baikalospongia intermedia was evaluated by sequence analysis of 16S rRNA genes from two sponge samples collected in Lake Baikal from depths of 550 and 1204 m. A total of 64 operational taxonomic units, belonging to nine bacterial phyla, Proteobacteria (classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria), Actinobacteria, Planctomycetes, Cloroflexi, Verrucomicrobia, Acidobacteria, Chlorobi, and Nitrospirae, including candidate phylum WS5, were identified. Phylogenetic analysis showed that the examined communities contained phylotypes exhibiting homology to uncultured bacteria from different lake ecosystems, freshwater sediments, soil and geological formations. Moreover, a number of phylotypes were relative to psychrophilic, methane-oxidizing, sulfate-reducing bacteria, and to microorganisms resistant to the influence of heavy metals. It is noted that the unusual habitation conditions of deep-water sponges contribute to the taxonomic diversity of associated bacteria and have an influence on the presence of functionally important microorganisms in bacterial communities.     

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.     

Bacterial community in the rhizosphere of the cactus species Mammillaria carnea during dry and rainy seasons assessed by deep sequencing

Background and aims

The Tehuacán-Cuitcatlán reserve is an area of unique plant biodiversity mostly in the form of xerophytes, with exceptionally high numbers of rare and endemic species. This endemism results partly from the characteristics of the climate of this area, with two distinct seasons: rainy and dry seasons. Although rhizosphere communities must be critical in the function of this ecosystem, understanding the structure of these communities is currently limited. This is the first molecular study of the microbial diversity present in the rhizosphere of Mamillaria carnea.

Methods

Total DNA was obtained from soil and rhizosphere samples at three locations in the Tehuacán Cuicatlán Reserve, during dry and rainy seasons. Temperature gradient gel electrophoresisis (TGGE) fingerprinting, 16S rRNA gene libraries and pyrosequencing were used to investigate bacterial diversity in the rhizosphere of Mammillaria carnea and changes in the microbial community between seasons.

Results

Deep sequencing data reveal a higher level of biodiversity in the dry season. Statistical analyses based on these data indicates that the composition of the bacterial community differed between both seasons affecting to members of the phyla Acidobacteria, Cyanobacteria, Gemmatimonadetes, Plantomycetes, Actinobacteria and Firmicutes. In addition, the depth of sequencing performed (>24,000 reads) enables detection of changes in the relative abundance of lower bacterial taxa (novel bacterial phylotypes) indicative of the increase of specific bacterial populations due to the season.

Conclusions

This study states the basis of the bacterial diversity in the rhizosphere of cacti in semi-arid environments and it is a sequence-based demonstration of community shifts in different seasons.     

16S rRNA gene analyses of bacterial community structures in the soils of evergreen broad-leaved forests in south-west China

Bacterial community structure was studied in humus and mineral soils of evergreen broad-leaved forests in Ailaoshan and Xishuangbanna, representing subtropical and tropical ecosystems, respectively, in south-west China using sequence analysis and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. Clone sequences affiliated to Acidobacteria were retrieved as the predominant bacterial phylum in both forest soils, followed by those affiliated to members of the Proteobacteria, Planctomycete and Verrucomicrobia. Despite higher floristic richness at the Xishuangbanna forest than at the Ailaoshan forest, soil at Xishuangbanna harbored a distinctly high relative abundance of Acidobacteria-affiliated sequences (80% of the total clones), which led to a lower overall bacterial diversity than at Ailaoshan. Bacterial communities in humus and mineral soils of the two forests appeared to be well differentiated, based on 16S rRNA gene phylogeny, and correlations were found between the bacterial T-RFLP community patterns and the organic carbon and nutrient contents of the soil samples. The data reveal that Acidobacteria dominate soil bacterial communities in the evergreen broad-leaved forests studied here and suggest that bacterial diversity may be influenced by soil carbon and nutrient levels, but is not related to floristic richness along the climatic gradient from subtropical to tropical forests in south-west China.     

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.     

Bacterial community structure in the rhizosphere of three cactus species from semi-arid highlands in central Mexico

The nature reserve of Tehuacan-Cuicatlan in central Mexico is known for its diversity and endemism mainly in cactus plants. Although the xerophytic flora is reasonably documented, the bacterial communities associated with these species have been largely neglected. We assessed the diversity and composition of bacterial communities in bulk (non-rhizospheric) soil and the rhizosphere of three cactus plant species: Mammillaria carnea, Opuntia pilifera and Stenocereus stellatus, approached using cultivation and molecular techniques, considering the possible effect of dry and rainy seasons. Cultivation-dependent methods were focused on putative N₂-fixers and heterotrophic aerobic bacteria, in the two media tested the values obtained for dry season samples grouped together regardless of the sample type (rhizospheric or non-rhizospheric), these groups also included the non-rhizospheric sample for rainy season, on each medium. These CFU values were smaller and significantly different from those obtained on rhizospheric samples from rainy season. Genera composition among isolates of the rhizospheric samples was very similar for each season, the most abundant taxa being α-Proteobacteria, Actinobacteria and Firmicutes. Interestingly, the genus Ochrobactrum was highly represented among rhizospheric samples, when cultured in N-free medium. The structure of the bacterial communities was approached with molecular techniques targeting partial 16S rRNA sequences such as denaturing gradient gel electrophoresis and serial analysis of ribosomal sequence tags. Under these approaches, the most represented bacterial phyla were Actinobacteria, Proteobacteria and Acidobacteria. The first two were also highly represented when using isolation techniques.     

Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods

A combination of culture-dependent and culture-independent methodologies (Bacteria and Archaea 16S rRNA gene clone library analyses) was used to determine the microbial diversity present within a geographically distinct high Arctic permafrost sample. Culturable Bacteria isolates, identified by 16S rRNA gene sequencing, belonged to the phyla Firmicutes, Actinobacteria and Proteobacteria with spore-forming Firmicutes being the most abundant; the majority of the isolates (19/23) were psychrotolerant, some (11/23) were halotolerant, and three isolates grew at -5 degrees C. A Bacteria 16S rRNA gene library containing 101 clones was composed of 42 phylotypes related to diverse phylogenetic groups including the Actinobacteria, Proteobacteria, Firmicutes, Cytophaga - Flavobacteria - Bacteroides, Planctomyces and Gemmatimonadetes; the bacterial 16S rRNA gene phylotypes were dominated by Actinobacteria- and Proteobacteria-related sequences. An Archaea 16S rRNA gene clone library containing 56 clones was made up of 11 phylotypes and contained sequences related to both of the major Archaea domains (Euryarchaeota and Crenarchaeota); the majority of sequences in the Archaea library were related to halophilic Archaea. Characterization of the microbial diversity existing within permafrost environments is important as it will lead to a better understanding of how microorganisms function and survive in such extreme cryoenvironments.