Identification and Isolation of a Castellaniella Species Important during Biostimulation of an Acidic Nitrate- and Uranium-Contaminated Aquifer

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.     

Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.     

九龙江河口区nirS型反硝化细菌多样性及系统发育学分析

【目的】结合16S rRNA基因克隆文库和nirS基因克隆文库的分析,揭示九龙江河口区nirS型反硝化细菌多样性。【方法】选取九龙江河口区一富营养化采样点,分别采集水样及沉积物样品,进行理化因子的测定并提取细菌总DNA。以水样DNA构建16S rRNA基因克隆文库,以沉积物DNA构建nirS基因克隆文库,分析微生物群落结构的多样性并构建系统发育树。【结果】从16S rRNA基因克隆文库中获得86条有效序列,按97%的序列相似性划分为53个OTU,分别属于Proteobacteria门、Planctomycetes门、Bacteroidetes门、Actinobacteria门、Firmicutes门和Chloroflexi门。其中属于Proteobacteria门OTU的克隆子占克隆数的62.9%,是最优势的类群,分属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Deltaproteobacteria纲等。从nirS基因克隆文库中获得190条有效序列,翻译为氨基酸序列后,按82%的序列相似性划分为60个OTU,并定位到属的水平。其中Proteobacteria门是最优势的类群,占文库克隆子总数的71.6%,包括Alphaproteobacteria纲(5.8%)、Betaproteobacteria纲(49.0%)和Gammaproteobacteria纲(16.9%)。nirS基因克隆文库中丰度最高的OTU与GenBank中的一株可培养反硝化菌Thauera sp. R-26906具有100%的序列相似性。【结论】九龙江河口区的微生物以及亚硝酸盐还原酶基因(nirS)具有丰富的多样性。大部分NirS序列在GenBank中的最相似序列来源于河口、海湾等相似的环境。     

Diversity of nitrite reductase (nirK and nirS) gene fragments in forested upland and wetland soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers

Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed on cultivated denitrifiers to assess their incidence in different bacterial taxa and their taxonomical value. Almost half of the 227 investigated denitrifying strains did not render an nir amplicon with any of five previously described primers. NirK and nirS were found to be prevalent in Alphaproteobacteria and Betaproteobacteria, respectively, nirK was detected in the Firmicutes and Bacteroidetes and nirS and nirK with equal frequency in the Gammaproteobacteria. These observations deviated from the hitherto reported incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S rRNA gene phylogeny on family or genus level, although some strains did group within clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly more similar to nirK sequences from the same habitat than to nirK sequences retrieved from highly related taxa. This study supports the hypothesis that horizontal gene transfer events of denitrification genes have occurred and underlines that denitrification genes should not be linked with organism diversity of denitrifiers in cultivation-independent studies.     

Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.     

Change in bacterial community structure during in situ biostimulation of subsurface sediment cocontaminated with uranium and nitrate

Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater. Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells. In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed. Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted. A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the alpha, beta, delta, and gamma subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species. Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing delta-Proteobacteria increased from 5% to nearly 40% of the clone libraries. Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested. Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the delta-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments. This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment.     

Molecular diversity of denitrifying genes in continental margin sediments within the oxygen-deficient zone off the Pacific coast of Mexico

To understand the composition and structure of denitrifying communities in the oxygen-deficient zone off the Pacific coast of Mexico, the molecular diversity of nir genes from sediments obtained at four stations was examined by using a PCR-based cloning approach. A total of 50 operational taxonomic units (OTUs) for nirK and 82 OTUs for nirS were obtained from all samples. Forty-four of the nirS clones and 31 of the nirK clones were sequenced; the levels of similarity of the nirS clones were 52 to 92%, and the levels of similarity of the nirS clones were 50 to 99%. The percentages of overlapping OTUs between stations were 18 to 30% for nirS and 5 to 8% for nirK. Sequence analysis revealed that 26% of the nirS clones were related to the nirS genes of Alcaligenes faecalis (80 to 94% similar) and Pseudomonas stutzeri (80 to 99%), whereas 3 to 31% of the nirK clones were closely related to the nirK genes of Pseudomonas sp. strain G-179 (98 to 99%), Bradyrhizobium japonicum (91%), Blastobacter denitrificans (83%), and Alcaligenes xylosoxidans (96%). The rest of the clones, however, were less than 80% similar to nirS and nirK sequences available in sequence databases. The results of a principal-component analysis (PCA) based on the percentage of OTUs and biogeochemical data indicated that the nitrate concentration and oxygen have an effect on the denitrifying communities. The communities at the stations in oxygen-deficient zones were more similar than the communities at the stations in the oxygenated zone. The denitrifying communities were more similar at the stations that were closer together and had similar nitrate levels. Also, the results of PCA based on biogeochemical properties suggest that geographic location and biogeochemical conditions, especially the nitrate and oxygen levels, appear to be the key factors that control the structure of denitrifying communities.     

Molecular diversity and characterization of nitrite reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater

Nitrate-contaminated groundwater samples were analysed for nirK and nirS gene diversity. The samples differed with respect to nitrate, uranium, heavy metals, organic carbon content, pH and dissolved oxygen levels. A total of 958 nirK and 1162 nirS clones were screened by restriction fragment length polymorphism (RFLP) analysis: 48 and 143 distinct nirK and nirS clones, respectively, were obtained. A single dominant nirK restriction pattern was observed for all six samples and was 83% identical to the Hyphomicrobium zavarzinii nirK gene. A dominant nirS pattern was observed for four of the samples, including the background sample, and was 95% identical to the nirS of Alcaligenes faecalis. Diversity indices for nirK and nirS sequences were not related to any single geochemical characteristic, but results suggested that the diversity of nirK genes was inversely proportional to the diversity of nirS. Principal component analysis (PCA) of the sites based on geochemistry grouped the samples by low, moderate and high nitrate but PCA of the unique operational taxonomic units (OTUs) distributions grouped the samples differently. Many of the sequences were not closely related to previously observed genes and some phylogenetically related sequences were obtained from similar samples. The results indicated that the contaminated groundwater contained novel nirK and nirS sequences, functional diversity of both genes changed in relation to the contaminant gradient, but the nirK and nirS functional diversity was affected differently.     

Environmental whole-genome amplification to access microbial populations in contaminated sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.     

Temporal and spatial variability in nearshore bacterioplankton communities of Lake Michigan

The spatial and temporal variability of bacterial communities were determined for the nearshore waters of Lake Michigan, an oligotrophic freshwater inland sea. A freshwater estuary and nearshore sites were compared six times during 2006 using denaturing gradient gel electrophoresis (DGGE). Bacterial composition clustered by individual site and date rather than by depth. Seven 16S rRNA gene clone libraries were constructed, yielding 2717 bacterial sequences. Spatial variability was detected among the DGGE banding patterns and supported by clone library composition. The clone libraries from deep waters and the estuary environment revealed highest overall bacterial diversity. Betaproteobacteria sequence types were the most dominant taxa, comprising 40.2–67.7% of the clone libraries. BAL 47 was the most abundant freshwater cluster of Betaproteobacteria , indicating widespread distribution of this cluster in the nearshore waters of Lake Michigan. Incertae sedis 5 and Oxalobacteraceae sequence types were prevalent in each clone library, displaying more diversity than previously described in other freshwater environments. Among the Oxalobacteraceae sequences, a globally distributed freshwater cluster was determined. The nearshore waters of Lake Michigan are a dynamic environment that experience forces similar to the coastal ocean environment and share common bacterial diversity with other freshwater habitats.     

Analysis of nitrite reductase (nirK and nirS) genes and cultivation reveal depauperate community of denitrifying bacteria in the Black Sea suboxic zone

Chemical profiles of the Black Sea suboxic zone show a distribution of nitrogen species which is traditionally associated with denitrification, i.e. a secondary nitrite maximum associated with nitrate depletion and a N(2) gas peak. To better understand the distribution and diversity of the denitrifier community in the Black Sea suboxic zone, we combined a cultivation approach with cloning and sequencing of PCR-amplified nitrite reductase (nirS and nirK) genes. The Black Sea suboxic zone appears to harbour a homogeneous community of denitrifiers. For nirK, over 94% of the sequences fell into only three distinct phylogenetic clusters, and for nirS, a single closely related sequence type accounted for 91% of the sequences retrieved. Both nirS and nirK genes showed a dramatic shift in community composition at the bottom of the suboxic zone, but overall, nirK-based community composition showed much greater variation across depths compared with the highly uniform distribution of nirS sequences throughout the suboxic zone. The dominant nirK and nirS sequences differed at the amino acid level by at least 17% and 8%, respectively, from their nearest database matches. Denitrifying isolates recovered from the suboxic zone shared 97% 16S rRNA gene sequence similarity with Marinobacter maritimus. Analysis of the recently discovered nirS gene from the anammox bacterium Candidatus'Kuenenia stuttgartiensis' revealed that mismatches with commonly used primers may have prevented the previous detection of this divergent sequence.     

Denitrifier community composition along a nitrate and salinity gradient in a coastal aquifer

Nitrogen flux into the coastal environment via submarine groundwater discharge may be modulated by microbial processes such as denitrification, but the spatial scales at which microbial communities act and vary are not well understood. In this study, we examined the denitrifying community within the beach aquifer at Huntington Beach, California, where high-nitrate groundwater is a persistent feature. Nitrite reductase-encoding gene fragments (nirK and nirS), responsible for the key step in the denitrification pathway, were PCR amplified, cloned, and sequenced from DNAs extracted from aquifer sediments collected along a cross-shore transect, where groundwater ranged in salinity from 8 to 34 practical salinity units and in nitrate concentration from 0.5 to 330 muM. We found taxonomically rich and novel communities, with all nirK clones exhibiting <85% identity and nirS clones exhibiting <92% identity at the amino acid level to those of cultivated denitrifiers and other environmental clones in the database. Unique communities were found at each site, despite being located within 40 m of each other, suggesting that the spatial scale at which denitrifier diversity and community composition vary is small. Statistical analyses of nir sequences using the Monte Carlo-based program integral-Libshuff confirmed that some populations were indeed distinct, although further sequencing would be required to fully characterize the highly diverse denitrifying communities at this site.     

晋南牛瘤胃中古菌分子多样性的研究

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立克隆库来研究晋南牛瘤胃古菌的多样性.每个克隆库随机挑选100个克隆.引物Arch f364/1386建立的克隆库中,克隆分为四类,分别与四种甲烷短杆菌1Y(61%)、SM9(23%)、NT7(14%)和AK-87(2%)相似.引物1Af/1100Ar建立的克隆库中,克隆分为两类,分别与Methanobacterium aarhusense(72%)和Methanosphaera stadtmanae DSM 3091(28%)相似.引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面,除以上4种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外,还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列,还有7%的未匹配序列.系统进化分析表明,这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium,和未知广域古菌等5个分支.有25类属于广域古菌的未知序列,提示瘤胃中存在大量的未知产甲烷菌.     

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.     

Novel eukaryotic lineages inferred from small-subunit rRNA analyses of oxygen-depleted marine environments

Microeukaryotes in oxygen-depleted environments are among the most diverse, as well as the least studied, organisms. We conducted a cultivation-independent, small-subunit (SSU) rRNA-based survey of microeukaryotes in suboxic waters and anoxic sediments in the great Sippewisset salt marsh, Cape Cod, Mass. We generated two clone libraries and analyzed approximately 300 clones, which contained a large diversity of microeukaryotic SSU rRNA signatures. Only a few of these signatures were closely related (sequence similarity of >97%) to the sequences reported earlier. The bulk of our sequences represented deep novel branches within green algae, fungi, cercozoa, stramenopiles, alveolates, euglenozoa and unclassified flagellates. In addition, a significant number of detected rRNA sequences exhibited no affiliation to known organisms and sequences and thus represent novel lineages of the highest taxonomical order, most of them branching off the base of the global phylogenetic tree. This suggests that oxygen-depleted environments harbor diverse communities of novel organisms, which may provide an interesting window into the early evolution of eukaryotes.