Metabolically active microbial communities in uranium-contaminated subsurface sediments

In order to develop effective bioremediation strategies for radionuclide contaminants, the composition and metabolic potential of microbial communities need to be better understood, especially in highly contaminated subsurface sediments for which little cultivation-independent information is available. In this study, we characterized metabolically active and total microbial communities associated with uranium-contaminated subsurface sediments along geochemical gradients. DNA and RNA were extracted and amplified from four sediment-depth intervals representing moderately acidic (pH 3.7) to near-neutral (pH 6.7) conditions. Phylotypes related to Proteobacteria (Alpha-, Beta-, Delta- and Gammaproteobacteria), Bacteroidetes, Actinobacteria, Firmicutes and Planctomycetes were detected in DNA- and RNA-derived clone libraries. Diversity and numerical dominance of phylotypes were observed to correspond to changes in sediment geochemistry and rates of microbial activity, suggesting that geochemical conditions have selected for well-adapted taxa. Sequences closely related to nitrate-reducing bacteria represented 28% and 43% of clones from the total and metabolically active fractions of the microbial community, respectively. This study provides the first detailed analysis of total and metabolically active microbial communities in radionuclide-contaminated subsurface sediments. Our microbial community analysis, in conjunction with rates of microbial activity, points to several groups of nitrate-reducers that appear to be well adapted to environmental conditions common to radionuclide-contaminated sites.     

Multiple influences of nitrate on uranium solubility during bioremediation of uranium-contaminated subsurface sediments

Microbiological reduction of soluble U(VI) to insoluble U(IV) has been proposed as a remediation strategy for uranium-contaminated groundwater. Nitrate is a common co-contaminant with uranium. Nitrate inhibited U(VI) reduction in acetate-amended aquifer sediments collected from a uranium-contaminated site in New Mexico. Once nitrate was depleted, both U(VI) and Fe(III) were reduced concurrently. When nitrate was added to sediments in which U(VI) had been reduced, U(VI) reappeared in solution. Parallel studies with the dissimilatory Fe(III)-, U(VI)- and nitrate-reducing microorganism, Geobacter metallireducens, demonstrated that nitrate inhibited reduction of Fe(III) and U(VI) in cell suspensions of cells that had been grown with nitrate as the electron acceptor, but not in Fe(III)-grown cells. Suspensions of nitrate-grown G. metallireducens oxidized Fe(II) and U(IV) with nitrate as the electron acceptor. U(IV) oxidation was accelerated when Fe(II) was also added, presumably due to the Fe(III) being formed abiotically oxidizing U(IV). These studies demonstrate that although the presence of nitrate is not likely to be an impediment to the bioremediation of uranium contamination with microbial U(VI) reduction, it is necessary to reduce nitrate before U(VI) can be reduced. These results also suggest that anaerobic oxidation of U(IV) to U(VI) with nitrate serving as the electron acceptor may provide a novel strategy for solubilizing and extracting microbial U(IV) precipitates from the subsurface.     

Group-specific PCR primers for the phylum Acidobacteria designed based on the comparative analysis of 16S rRNA gene sequences

We performed a comprehensive phylogenetic analysis of the phylum Acidobacteria and developed novel, group-specific PCR primers for Acidobacteria and its class-level subgroups. Acidobacterial 16S rRNA gene sequences deposited in the RDP database were used to construct a local database then subsequently analyzed. A total of 556 phylotypes were observed and the majority of the phylotypes belonged to five major subgroups (subgroups 1, 2, 3, 4, and 6), which comprised > 80% of the acidobacterial sequences in the RDP database. Phylum-specific and subgroup-specific primers were designed from the consensus sequences of the phylotype sequences, and the specificities of the designed primers were evaluated both in silico and empirically for coverage and tolerance. The phylum-specific primer ACIDO, which was designed in this study, showed increased coverage for Acidobacteria, as compared to the previous phylum-specific primer 31F. However, the tolerance of the primer ACIDO for non-target sequences was slightly higher than that of the primer 31F. We also developed subgroup-specific PCR primers for the major subgroups of Acidobacteria, except for subgroup 4. Subgroup-specific primers S1, S2, and S3, which targeted subgroups 1, 2, and 3, respectively, showed high coverage for their target subgroups and low tolerance for non-target sequences. However, the primer S6 targeting subgroup 6 showed a lower specificity in its empirical evaluation than expected from the in silico results. The subgroup-specific primers, as well as the phylum-specific primer designed in this study, will be valuable tools in understanding the phylogenetic diversity and ecological niche of the phylum Acidobacteria and its subgroups.     

Deep biosphere-related bacteria within the subsurface of tidal flat sediments

Biogeochemical and microbiological processes in the upper sediment layers of tidal flats were analysed in many investigations, while deeper zones remained largely unexplored. Therefore, denaturant gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments along the depth profile of up to 5.5 m-long sediment cores was performed in comparison with lithological and geochemical parameters. The investigation revealed that different compartments of the sediment columns were characterized by specific microbial communities. These compartments were analysed by sequencing of 113 DGGE bands. The upper layers down to 160-200 cm were dominated by gamma- and delta-Proteobacteria representing more than 60% of the total number of phylotypes. Underneath, a striking shift in community composition was observed, as the Proteobacteria were replaced by Chloroflexi with more than 60% of all sequences. As sulfate was still available as an electron acceptor in these layers, the abundance of Chloroflexi might be promoted by the electron donor or the quality of the carbon source. The dominance of this group, previously known as green non-sulfur bacteria, indicates the presence of a typical deep-biosphere microbial community in relatively young subsurface sediments. Thus, tidal flats might offer a convenient possibility to study and understand certain aspects of the deep biosphere in general.     

Metagenomic analysis reveals unexpected subgenomic diversity of magnetotactic bacteria within the phylum Nitrospirae

A targeted metagenomic approach was applied to investigate magnetotactic bacteria (MTB) within the phylum Nitrospirae in Lake Miyun near Beijing, China. Five fosmids containing rRNA operons were identified. Comparative sequence analysis of a total of 172 kb provided new insights into their genome organization and revealed unexpected subgenomic diversity of uncultivated MTB in the phylum Nitrospirae. In addition, affiliation of two novel MTB with the phylum Nitrospirae was verified by fluorescence in situ hybridization. One of them was morphologically similar to "Candidatus Magnetobacterium bavaricum," but the other differed substantially in cell shape and magnetosome organization from all previously described "Ca. Magnetobacterium bavaricum"-like bacteria.     

Influence of plant polymers on the distribution and cultivation of bacteria in the phylum Acidobacteria

Members of the phylum Acidobacteria are among the most abundant bacteria in soil. Although they have been characterized as versatile heterotrophs, it is unclear if the types and availability of organic resources influence their distribution in soil. The potential for organic resources to select for different acidobacteria was assessed using molecular and cultivation-based approaches with agricultural and managed grassland soils in Michigan. The distribution of acidobacteria varied with the carbon content of soil: the proportion of subdivision 4 sequences was highest in agricultural soils (ca. 41%) that contained less carbon than grassland soils, where the proportions of subdivision 1, 3, 4, and 6 sequences were similar. Either readily oxidizable carbon or plant polymers were used as the sole carbon and energy source to isolate heterotrophic bacteria from these soils. Plant polymers increased the diversity of acidobacteria cultivated but decreased the total number of heterotrophs recovered compared to readily oxidizable carbon. Two phylogenetically novel Acidobacteria strains isolated on the plant polymer medium were characterized. Strains KBS 83 (subdivision 1) and KBS 96 (subdivision 3) are moderate acidophiles with pH optima of 5.0 and 6.0, respectively. Both strains grew slowly (μ = 0.01 h(-1)) and harbored either 1 (strain KBS 83) or 2 (strain KBS 96) copies of the 16S rRNA encoding gene-a genomic characteristic typical of oligotrophs. Strain KBS 83 is a microaerophile, growing optimally at 8% oxygen. These metabolic characteristics help delineate the niches that acidobacteria occupy in soil and are consistent with their widespread distribution and abundance.     

Denitrification in deep subsurface sediments

Dissimilatory nitrate reduction (denitrification) in subsurface sediments by indigenous microflora was investigated in samples obtained over a range of depths from 0 to 289 m. Denitrifying activity in sediment samples retrieved from similar stratigraphic horizons at four different sites was determined by measuring the accumulation of N₂O using the acetylene blockage technique. Denitrification was detected in unamended samples which received only prereduced deionized water at almost all depths in all sediments sampled. The surface sediments showed the highest denitrification activity. In the deeper sediments, denitrifying activity was much higher in saturated sandy samples and lower or absent in drier clay samples. Addition of nitrate enhanced denitrification activity in all samples from below the water table down to the maximum depth sampled (289 m), while addition of a carbon (succinate) source in general had no stimulatory effect. These results show that denitrifying microorganisms were present in all of the deep subsurface sediments tested in this study. Furthermore, these results suggest that adequate supplies of metabolizable organic carbon were available to support denitrifying activity. However, denitrification may be limited by inadequate supplies of nitrate in the sediments.     

Analysis of environmental 18S ribosomal RNA sequences reveals unknown diversity of the cosmopolitan phylum Telonemia

Telonemia has recently been described as a new eukaryotic phylum with uncertain evolutionary origin. So far, only two Telonemia species, Telonema subtilis and Telonema antarcticum, have been described, but there are substantial variations in size and morphology among Telonema isolates and field observations, indicating a hidden diversity of Telonemia-like species and populations. In this study, we investigated the diversity and the global distribution of this group by analyzing 18S rDNA sequences from marine environmental clone libraries published in GenBank as well as several unpublished sequences from the Indian Ocean. Phylogenetic analyses of the identified sequences suggest that the Telonemia phylum includes several undescribed 18S rDNA phylotypes, probably corresponding to a number of different species and/or populations. The Telonemia phylotypes form two main groups, here referred to as Telonemia Groups 1 and 2. Some of the closely related sequences originate from separate oceans, indicating worldwide distributions of various Telonemia phylotypes, while other phylotypes seem to have limited geographical distribution. Further investigations of the evolutionary relationships within Telonemia should be conducted on isolated cultures of Telonema-like strains using multi-locus sequencing and morphological data.     

Widespread distribution and high abundance of Rhizobium radiobacter within Mediterranean subsurface sediments

Eastern Mediterranean sediments are characterized by the occurrence of distinct, organic-rich layers, called sapropels. These harbour elevated microbial numbers in comparison with adjacent carbon-lean intermediate layers. A recently obtained culture collection from these sediments was composed of 20% of strains closely related to Rhizobium radiobacter, formerly classified as Agrobacterium tumefaciens. To prove and quantify the in situ abundance of R. radiobacter, a highly specific quantitative polymerase chain reaction (PCR) protocol was developed. To convert quantification results into cell numbers, the copy number of rrn operons per genome was determined. Southern hybridization showed that our isolates contained four operons. Finally, quantitative PCR was applied to 45 sediment samples obtained across the eastern Mediterranean. Rhizobium radiobacter was present in 38 of 45 samples indicating an almost ubiquitous distribution. In total, 25-40 000 cells per gram of sediment were detected, corresponding to 0.001-5.1% of the bacterial cells. In general, the relative and absolute abundance of R. radiobacter increased with depth and was higher in sapropels than in intermediate layers. This indicates that R. radiobacter forms an active population in up to 200 000 years old sapropels. The present study shows for the first time that a cultivated subsurface bacterium is highly abundant in this environment.     

Complete genome of Candidatus Chloracidobacterium thermophilum, a chlorophyll-based photoheterotroph belonging to the phylum Acidobacteria

Candidatus Chloracidobacterium thermophilum, which naturally inhabits microbial mats of alkaline siliceous hot springs in Yellowstone National Park, is the only known chlorophototroph in the phylum Acidobacteria. The Ca. C. thermophilum genome was composed of two chromosomes (2,683,362 bp and 1,012,010 bp), and both encoded essential genes. The genome included genes to produce chlorosomes, the Fenna-Matthews-Olson protein, bacteriochlorophylls a and c as principal pigments, and type-1, homodimeric reaction centres. Ca. C. thermophilum is an aerobic photoheterotroph that lacks the ability to synthesize several essential nutrients. Key genes of all known carbon fixation pathways were absent, as were genes for assimilatory nitrate and sulfate reduction and vitamin B(12) synthesis. Genes for the synthesis of branched-chain amino acids (valine, isoleucine and leucine) were also absent, but genes for catabolism of these compounds were present. This observation suggested that Ca. C. thermophilum may synthesize branched-chain amino acids from an intermediate(s) of the catabolic pathway by reversing these reactions. The genome encoded an aerobic respiratory electron transport chain that included NADH dehydrogenase, alternative complex III and cytochrome oxidase. The chromosomes of the laboratory isolate were compared with assembled, metagenomic scaffolds from the major Ca. C. thermophilum population in hot-spring mats. The larger chromosomes of the two populations were highly syntenous but significantly divergent (~13%) in sequence. In contrast, the smaller chromosomes have undergone numerous rearrangements, contained many transposases, and might be less constrained by purifying selection than the large chromosomes. Some transposases were homologous to those of mat community members from other phyla.     

Isolation and characterization of soil bacteria that define Terriglobus gen. nov., in the phylum Acidobacteria

Bacteria in the phylum Acidobacteria are widely distributed and abundant in soils, but their ecological roles are poorly understood, owing in part to a paucity of cultured representatives. In a molecular survey of acidobacterial diversity at the Michigan State University Kellogg Biological Station Long-Term Ecological Research site, 27% of acidobacterial 16S rRNA gene clones in a never-tilled, successional plant community belonged to subdivision 1, whose relative abundance varied inversely with soil pH. Strains of subdivision 1 were isolated from these never-tilled soils using low-nutrient medium incubated for 3 to 4 weeks under elevated levels of carbon dioxide, which resulted in a slightly acidified medium that matched the pH optima of the strains (between 5 and 6). Colonies were approximately 1 mm in diameter and either white or pink, the latter due to a carotenoid(s) that was synthesized preferentially under 20% instead of 2% oxygen. Strains were gram-negative, aerobic, chemo-organotrophic, nonmotile rods that produced an extracellular matrix. All strains contained either one or two copies of the 16S rRNA encoding gene, which along with a relatively slow doubling time (10 to 15 h at ca. 23 degrees C) is suggestive of an oligotrophic lifestyle. Six of the strains are sufficiently similar to one another, but distinct from previously named Acidobacteria, to warrant creation of a new genus, Terriglobus, with Terriglobus roseus defined as the type species. The physiological and nutritional characteristics of Terriglobus are consistent with its potential widespread distribution in soil.     

Phylogenetic relationships within the class Anthozoa (phylum Cnidaria) based on nuclear 18S rDNA sequences

Taxonomic relationships within the corals and anemones (Phylum Cnidaria: Class Anthozoa) are based upon few morphological characters. The significance of any given character is debatable, and there is little fossil record available for deriving evolutionary relationships. We analyzed complete 18S ribosomal sequences to examine subclass-level and ordinal-level organization within the Anthozoa. We suggest that the Subclass Ceriantipatharia is not an evolutionarily relevant grouping. The Order Corallimorpharia appears paraphyletic and closely related to the Order Scleractinia. The 18S rRNA gene may be insufficient for establishing robust phylogenetic hypotheses concerning the specific relationships of the Corallimorpharia and the Ceriantharia and the branching sequence for the orders within the Hexacorallia. The 18S rRNA gene has sufficient phylogenetic signal, however, to distinguish among the major groupings within the Class Anthozoa, and we use this information to suggest relationships for the enigmatic taxa Dactylanthus and Dendrobrachia.     

Reiterated DNA sequences defining genomic diversity within the species Mycoplasma hyorhinis

Genomic restriction fragments isolated from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae were shown by DNA hybridization and nucleotide sequence analyses to contain sequences common to these two species, as well as another porcine-derived mycoplasma, Mycoplasma flocculare. Intraspecies hybridization experiments using these fragments as probes indicated that the sequence is highly redundant in several strains of M. hyorhinis, but that there is diversity in the sizes of restriction fragments detected among these strains. In contrast, repetition of the sequence was limited in M. hyopneumoniae and M. flocculare, and no homologies to this repeated element were apparent in mycoplasma species isolated from animal hosts other than the swine. The reiterated sequence may reflect intraspecies genomic diversification in M. hyorhinis and its selective presence in otherwise unrelated species raises the possibility that it has been horizontally transmitted between these organisms.     

Thermotomaculum hydrothermale gen. nov., sp. nov., a novel heterotrophic thermophile within the phylum Acidobacteria from a deep-sea hydrothermal vent chimney in the Southern Okinawa Trough

A novel heterotrophic, thermophilic bacterium, designated strain AC55^T, was isolated from a deep-sea hydrothermal vent chimney at the Hatoma Knoll in the Okinawa Trough, Japan. Cells of strain AC55^T were non-motile, long rods (2.0- to 6.8-μm long and 0.3- to 0.6-μm wide). The strain was an obligatory anaerobic heterotroph capable of fermentative growth on complex proteinaceous substances. Elemental sulfur was reduced to hydrogen sulfide but did not stimulate growth. Growth was observed between 37 and 60°C (optimum 55°C), pH 5.5 and 8.5 (optimum pH 6.6), and in the presence of 1.5–4.5% (w/v) NaCl (optimum 2.5%, w/v). Menaquinone-7 and -8 were the major respiratory quinones. The G + C content of the genomic DNA from strain AC55^T was 51.6 mol%. The 16S rRNA gene sequence analysis revealed that strain AC55^T was the first cultivated representative of Acidobacteria subdivision 10. Based on the physiological and phylogenetic features of the novel isolate, the genus name Thermotomaculum gen. nov. is proposed, with Thermotomaculum hydrothermale sp. nov. as the type species. The type strain is AC55^T (=JCM 17643^T = DSM 24660^T = NBRC 107904^T).     

Effect of pH on isolation and distribution of members of subdivision 1 of the phylum Acidobacteria occurring in soil

The pH strongly influenced the development of colonies by members of subdivision 1 of the phylum Acidobacteria on solid laboratory media. Significantly more colonies of this group formed at pH 5.5 than at pH 7.0. At pH 5.5, 7 to 8% of colonies that formed on plates that were incubated for 4 months were formed by subdivision 1 acidobacteria. These colonies were formed by bacteria that spanned almost the entire phylogenetic breadth of the subdivision, and there was considerable congruence between the diversity of this group as determined by the cultivation-based method and by surveying 16S rRNA genes in the same soil. Members of subdivision 1 acidobacteria therefore appear to be readily culturable. An analysis of published libraries of 16S rRNAs or 16S rRNA genes showed a very strong correlation between the abundance of subdivision 1 acidobacteria in soil bacterial communities and the soil pH. Subdivision 1 acidobacteria were most abundant in libraries from soils with pHs of <6, but rare or absent in libraries from soils with pHs of >6.5. This, together with the selective cultivation of members of the group on lower-pH media, indicates that growth of many members of subdivision 1 acidobacteria is favored by slightly to moderately acidic growth conditions.     

Microbial diversity in coastal subsurface sediments: a cultivation approach using various electron acceptors and substrate gradients

Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, "Bacteroidetes," "Fusobacteria," Actinobacteria, and "Firmicutes." Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.