Spatial Heterogeneity of Crenarchaeal Assemblages within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling

Microbial ecologists have discovered novel rRNA genes (rDNA) in mesophilic soil habitats worldwide, including sequences that affiliate phylogenetically within the division Crenarchaeota (domain Archaea). To characterize the spatial distribution of crenarchaeal assemblages in mesophilic soil habitats, we profiled amplified crenarchaeal 16S rDNA sequences from diverse soil ecosystems by using PCR-single-stranded-conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP profiles provide a measure of relative microbial diversity in terms of richness (number of different phylotypes as estimated from the number of unique PCR-SSCP peaks) and evenness (abundance of each phylotype as estimated from the relative area under a peak). Crenarchaeal assemblages inhabiting prairie, forest, turf, and agricultural soils were characterized at six sampling locations in southern and central Wisconsin. Phylotype richness was found to be more stable than evenness among triplicate samples collected within 30 cm at each sampling location. Transformation of the PCR-SSCP data by principal-component analysis, followed by statistical testing (analysis of variance [P < 0.0001] and least-significant-difference analysis [α = 0.5]), supported the conclusion that each location exhibited a unique profile. To further characterize the spatial distribution of crenarchaeal assemblages at one location, additional soil samples (a total of 30) were collected from agricultural field plots at the Hancock Agricultural Research Station. PCR-SSCP revealed a patchy spatial distribution of crenarchaeal assemblages within and between these plots. This mosaic of crenarchaeal assemblages was characterized by differences in phylotype evenness that could not be correlated with horizontal distance (15 to 30 m) or with depth (0 to 20 cm below the surface). Crenarchaeal 16S rDNA clone libraries were produced and screened for unique SSCP peaks. Clones representing the dominant phylotypes at each location were identified, sequenced, and found to group phylogenetically with sequences in crenarchaeal clade C1b.     

Comparison of crenarchaeal consortia inhabiting the rhizosphere of diverse terrestrial plants with those in bulk soil in native environments

To explore whether the crenarchaeal consortium found in the rhizosphere is distinct from the assemblage of crenarchaeotes inhabiting bulk soil, PCR-single-stranded-conformation polymorphism (PCR-SSCP) profiles were generated for 76 plant samples collected from native environments. Divergent terrestrial plant groups including bryophytes (mosses), lycopods (club mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (seed plants) were collected for this study. Statistical analysis revealed significant differences between rhizosphere and bulk soil PCR-SSCP profiles (Hotelling paired T(2) test, P < 0.0001), suggesting that a distinct crenarchaeal consortium is associated with plants. In general, phylotype richness increased in the rhizosphere compared to the corresponding bulk soil, although the range of this increase was variable. Examples of a major change in rhizosphere (versus bulk soil) PCR-SSCP profiles were detected for all plant groups, suggesting that crenarchaeotes form associations with phylogenetically diverse plants in native environments. In addition, examples of minor to no detectable difference were found for all terrestrial plant groups, suggesting that crenarchaeal associations with plants are mediated by environmental conditions.     

Comparison of Crenarchaeal Consortia Inhabiting the Rhizosphere of Diverse Terrestrial Plants with Those in Bulk Soil in Native Environments

To explore whether the crenarchaeal consortium found in the rhizosphere is distinct from the assemblage of crenarchaeotes inhabiting bulk soil, PCR-single-stranded-conformation polymorphism (PCR-SSCP) profiles were generated for 76 plant samples collected from native environments. Divergent terrestrial plant groups including bryophytes (mosses), lycopods (club mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (seed plants) were collected for this study. Statistical analysis revealed significant differences between rhizosphere and bulk soil PCR-SSCP profiles (Hotelling paired T² test, P < 0.0001), suggesting that a distinct crenarchaeal consortium is associated with plants. In general, phylotype richness increased in the rhizosphere compared to the corresponding bulk soil, although the range of this increase was variable. Examples of a major change in rhizosphere (versus bulk soil) PCR-SSCP profiles were detected for all plant groups, suggesting that crenarchaeotes form associations with phylogenetically diverse plants in native environments. In addition, examples of minor to no detectable difference were found for all terrestrial plant groups, suggesting that crenarchaeal associations with plants are mediated by environmental conditions.     

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.     

DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil

A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82 degrees 30'N, 62 degrees 19'W). The enrichment culture was grown on Jet A-1 fuel at 7 degrees C. Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector. Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones. The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp. (six sequences), and Pseudomonas synxantha (four sequences). Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above. Phylotype-specific PCR assays were developed and validated for the above three phylotypes. The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays. Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype. A representative strain of each phylotype was physiologically characterized. Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes. During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay. All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library. The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium. The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations.     

Bacterial diversity and community structure in an aerated lagoon revealed by ribosomal intergenic spacer analyses and 16S ribosomal DNA sequencing

We investigated the bacterial community structure in an aerated plug-flow lagoon treating pulp and paper mill effluent. For this investigation, we developed a composite method based on analyses of PCR amplicons containing the ribosomal intergenic spacer (RIS) and its flanking partial 16S rRNA gene. Community percent similarity was determined on the basis of RIS length polymorphism. A community succession was evident in the lagoon, indicated by a progressive community transition through seven sample locations. The most abrupt changes in community structure were associated with a temperature change from 39 to 35 degrees C and with increases in dissolved oxygen. The temporal differences in community structure, based on summer and winter samplings, were greater than the spatial differences during either season. Clone libraries of rDNA-RIS amplicons were constructed from each of three summer samples. Among 90 clones analyzed (30 clones from each sample), 56 phylotypes were distinguished by restriction fragment length polymorphism. Indices of phylotype richness, evenness, and diversity all increased in clone libraries from the beginning to the end of the lagoon. A representative clone of each phylotype was phylogenetically analyzed on the basis of its partial 16S rRNA gene sequence (ca. 450 bp). Phylogenetic analysis confirmed the increase in diversity and further indicated increasing richness of bacterial divisions. Pioneers in the community spatial succession appeared to include thermotolerant, microaerophilic methanol-oxidizing bacteria related to the genus Methylobacillus, as well as thermotolerant, microaerophilic nitrogen-fixing bacteria related to the genus Azospirillum.     

Bacterial Diversity and Community Structure in an Aerated Lagoon Revealed by Ribosomal Intergenic Spacer Analyses and 16S Ribosomal DNA Sequencing

We investigated the bacterial community structure in an aerated plug-flow lagoon treating pulp and paper mill effluent. For this investigation, we developed a composite method based on analyses of PCR amplicons containing the ribosomal intergenic spacer (RIS) and its flanking partial 16S rRNA gene. Community percent similarity was determined on the basis of RIS length polymorphism. A community succession was evident in the lagoon, indicated by a progressive community transition through seven sample locations. The most abrupt changes in community structure were associated with a temperature change from 39 to 35°C and with increases in dissolved oxygen. The temporal differences in community structure, based on summer and winter samplings, were greater than the spatial differences during either season. Clone libraries of rDNA-RIS amplicons were constructed from each of three summer samples. Among 90 clones analyzed (30 clones from each sample), 56 phylotypes were distinguished by restriction fragment length polymorphism. Indices of phylotype richness, evenness, and diversity all increased in clone libraries from the beginning to the end of the lagoon. A representative clone of each phylotype was phylogenetically analyzed on the basis of its partial 16S rRNA gene sequence (ca. 450 bp). Phylogenetic analysis confirmed the increase in diversity and further indicated increasing richness of bacterial divisions. Pioneers in the community spatial succession appeared to include thermotolerant, microaerophilic methanol-oxidizing bacteria related to the genus Methylobacillus, as well as thermotolerant, microaerophilic nitrogen-fixing bacteria related to the genus Azospirillum.     

Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.     

Bacterial Succession in Glacial Forefield Soils Characterized by Community Structure,Activity and Opportunistic Growth Dynamics

The succession of bacterial communities inhabiting the forefield of the Dammaglacier (Switzerland) was investigated in soils ranging in successional age from 0 to 100 years since deglaciation. Overall activity per bacterial cell was estimated by the amount of fluorescein diacetate (FDA) hydrolyzed per DAPI-stained cell, and an index of "opportunism" was determined from the ratio of culturable to total cells (C:T ratio). Ribosomal intergenic spacer analysis (RISA) was used to estimate the richness of dominant phylotypes and to construct rank-abundance plots of the dominant populations. We observed a biphasic trend in specific cellular activity, which exhibited minima in the 0- and 100-year-old soils while a maximum activity per cell was reached in the 70-y soil. On average, the C:T ratio showed the same trend as the specific activity, although we observed some differences between the two sampling transects. RISA revealed a decrease in dominant phylotype richness as successional age increased, and rank-abundance plots indicated that the evenness of the dominant bacterial phylotypes significantly decreased with successional age. The combination of specific cellular activity and C:T ratio results suggested the presence of an r-K continuum of bacteria while RISA showed that richness and evenness of dominant phylotypes decreased with successional age. We conclude that bacterial succession in the glacier forefield was a dynamic process with adaptation to the differing stages of succession occurring on both the individual and community levels.     

Cultivation of Mesophilic Soil Crenarchaeotes in Enrichment Cultures from Plant Roots

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.     

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.     

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

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

Is evenness altered by fire in natural assemblages of soil arthropods?

We studied evenness and species richness in two assemblages of soil arthropods at six contiguous study plots in Mediterranean ecosystems of central Italy, three of these plots being burnt and three unburnt. We analysed these aspects of community structure by diversity–dominance diagrams comparisons made through analysis of covariance on respective slopes and ordinate intercepts. We observed consistent patterns in both Collembola and Oniscidea assemblages, either in burnt and unburnt plots. Evenness did not change among study plots and across habitats, either before or after fire, whereas species' composition was significantly altered by fire. Results from our study implied that evenness and species diversity are clearly affected in a different and independent way by fire. Hence, it is not acceptable to focus on only the evenness when looking at the effects of controlled fires for environmental management reasons.     

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

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

Biodiversity,community structural shifts,and biogeography of prokaryotes within Antarctic continental shelf sediment

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

Soil bacterial community shift correlated with change from forest to pasture vegetation in a tropical soil.

The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium-Agrobacterium assemblages in the pasture soil.     

Rapid change of AM fungal community in a rain-fed wheat field with short-term plastic film mulching practice

Plastic film mulching (PFM) is a widely used agricultural practice in the temperate semi-arid Loess Plateau of China. However, how beneficial soil microbes, arbuscular mycorrhizal (AM) fungi in particular, respond to the PFM practice is not known. Here, a field experiment was performed to study the effects of a 3-month short-term PFM practice on AM fungi in plots planted with spring wheat (Triticum aestivum L. cv. Dingxi-2) in the Loess Plateau. AM colonization, spore density, wheat spike weight, and grain phosphorus (P) content were significantly increased in the PFM treatments, and these changes were mainly attributable to changes in soil properties such as available P and soil moisture. Alkaline phosphatase activity was significantly higher in PFM soils, but levels of AM fungal-related glomalin were similar between treatments. A total of nine AM fungal phylotypes were detected in root samples based on AM fungal SSU rDNA analyses, with six and five phylotypes in PFM and no-PFM plots, respectively. Although AM fungal phylotype richness was not statistically different between treatments, the community compositions were different, with four and three specific phylotypes in the PFM and no-PFM plots, respectively. A significant and rapid change in AM fungal, wheat, and soil variables following PFM suggested that the functioning of the AM symbiosis had been changed in the wheat field under PFM. Future studies are needed to investigate whether PFM applied over a longer term has a similar effect on the AM fungal community and their functioning in an agricultural ecosystem.     

Diversity estimates of microeukaryotes below the chemocline of the anoxic Mariager Fjord, Denmark

Microbial communities of extreme environments have often been assumed to have low species richness. We analysed 18S rRNA gene signatures in a sample collected below the chemocline of the anoxic Mariager Fjord in Denmark, and from these data we computed novel parametric and standard nonparametric estimates of protistan phylotype richness. Our results indicate unexpectedly high richness in this environment: at the 99.5% phylotype definition, our most conservative estimate was 568 phylotypes (+/-114, standard error). Phylogenetic analyses revealed that the sequences collected cover the majority of described lineages in the eukaryotic domain. Out of 384 sequences analysed, 307 were identified as protistan targets, none of which was identical to known sequences. However, based on what is known about species that are phylogenetically related to the Mariager sequences, most of the latter seem to belong to strictly or facultative anaerobe organisms. We also found signatures that together with other environmental 18S rRNA gene sequences represent environmental clades of possibly high taxonomic levels (class to kingdom level). One of these clades, consisting exclusively of sequences from anoxic sampling sites, branches at the base of the eukaryotic evolutionary tree among the earliest eukaryotic lineages. Assuming eukaryotic evolution under oxygen-depleted conditions, these sequences may represent immediate descendants of early eukaryotic ancestors.