Diversity of 23S rRNA Genes within Individual Prokaryotic Genomes

Background

The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons.

Methodology/Principal Findings

Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4%) genomes (mean 0.40%, range 0.01%–4.04%). Significant (1.17%–4.04%) intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition). In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS), ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes.

Conclusions/Significance

These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.     

Molecular Phylogenetic and Biogeochemical Studies of Sulfate-Reducing Bacteria in the Rhizosphere of Spartina alterniflora

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO₄²⁻ reduction rates and geochemical parameters. SO₄²⁻ reduction was rapid in marsh sediments with rates up to 3.5 μmol ml⁻¹ day⁻¹. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of Bacteria rRNA did not vary greatly throughout the year, despite large temporal changes in SO₄²⁻ reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family Desulfobacteriaceae comprised the majority of the SRB rRNA at 3 to 34% of Bacteria rRNA, with Desulfobulbus spp. accounting for 1 to 16%. The RA of Desulfovibrio rRNA generally comprised from <1 to 3% of the Bacteria rRNA. The highest Desulfobacteriaceae RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10⁷ ml⁻¹. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of Bacteria rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.     

The genomic pattern of tDNA operon expression in E. coli

In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.     

Molecular Evolution of Mycoplasma capricolum subsp. capripneumoniae Strains, Based on Polymorphisms in the 16S rRNA Genes

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.     

Growth Rate Regulation of rRNA Content of a Marine Synechococcus (Cyanobacterium) Strain

The relationship between growth rate and rRNA content in a marine Synechococcus strain was examined. A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of Synechococcus strain WH8101 cells grown at a range of light-limited growth rates. The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing Synechococcus cells (μ = 0.15 day⁻¹). The relationship between growth rate and cellular rRNA content comprised three phases: (i) at low growth rates (<~0.7 day⁻¹), rRNA cell⁻¹ remained approximately constant; (ii) at intermediate rates (~0.7 − 1.6 day⁻¹), rRNA cell⁻¹ increased proportionally with growth rate; and (iii) at the highest, light-saturated rates (>~1.6 day⁻¹), rRNA cell⁻¹ dropped abruptly. Total cellular RNA (as measured with the nucleic acid stain SYBR Green II) was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate. Mean cell volume and rRNA concentration (amount of rRNA per cubic micrometer) were related to growth rate in a manner similar to rRNA cell⁻¹, although the overall magnitude of change in both cases was reduced. These patterns are hypothesized to reflect an approximately linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates. Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural Synechococcus populations.     

Quantification of Bacterial Groups within Human Fecal Flora by Oligonucleotide Probe Hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% ± 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% ± 7% and 14% ± 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans^T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae^T and Acidovorax facilis^T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10⁴ dimethylmalonate-utilizing cells ml⁻¹, representing up to 0.4% of the total culturable nitrate-reducing population.     

Community Structure, Cellular rRNA Content, and Activity of Sulfate-Reducing Bacteria in Marine Arctic Sediments

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell⁻¹ day⁻¹), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.     

A novel clade of Prochlorococcus found in high nutrient low chlorophyll waters in the South and Equatorial Pacific Ocean

A novel high-light (HL)-adapted Prochlorococcus clade was discovered in high nutrient and low chlorophyll (HNLC) waters in the South Pacific Ocean by phylogenetic analyses of 16S ribosomal RNA (rRNA) and 16S–23S internal transcribed spacer (ITS) sequences. This clade, named HNLC fell within the HL-adapted Prochlorococcus clade with sequences above 99% similarity to one another, and was divided into two subclades, HNLC1 and HNLC2. The distribution of the whole HNLC clade in a northwest to southeast transect in the South Pacific (HNLC-to-gyre) and two 8°N to 8°S transects in the Equatorial Pacific was determined by quantitative PCR using specific primers targeting ITS regions. HNLC was the dominant HL Prochlorococcus clade (2–9% of bacterial 16S rRNA genes) at the three westernmost stations in the South Pacific but decreased to less than 0.1% at the other stations being replaced by the eMIT9312 ecotype in the hyperoligotrophic gyre. The highest contributions of HNLC Prochlorococcus in both Equatorial Pacific transects along the latitudinal lines of 170°W and 155°W were observed at the southernmost stations, reaching 16 and 6% of bacterial 16S rRNA genes, respectively, whereas eMIT9312 dominated near the Equator. Spearman Rank Order correlation analysis indicated that although both the HNLC clade and eMIT9312 were correlated with temperature, they showed different correlations with regard to nutrients. HNLC only showed significant correlations to ammonium uptake and regeneration rates, whereas eMIT9312 was negatively correlated with inorganic nutrients.     

Quantitative Molecular Analysis of the Microbial Community in Marine Arctic Sediments (Svalbard)

Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI (4′,6′-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides δ-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the γ-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts, 14.4% ± 3.6% of prokaryotic rRNA). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts, 13.2% ± 4.6% of prokaryotic rRNA), showing no clear zonation along the vertical profile. Gram-positive bacteria and the β-proteobacteria were near the detection limit in all sediments.     

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.     

Insights into the metabolism,lifestyle and putative evolutionary history of the novel archaeal phylum ‘Diapherotrites'

The archaeal phylum ‘Diapherotrites'' was recently proposed based on phylogenomic analysis of genomes recovered from an underground water seep in an abandoned gold mine (Homestake mine in Lead, SD, USA). Here we present a detailed analysis of the metabolic capabilities and genomic features of three single amplified genomes (SAGs) belonging to the ‘Diapherotrites''. The most complete of the SAGs, Candidatus ‘Iainarchaeum andersonii'' (Cand. IA), had a small genome (∼1.24 Mb), short average gene length (822 bp), one ribosomal RNA operon, high coding density (∼90.4%), high percentage of overlapping genes (27.6%) and low incidence of gene duplication (2.16%). Cand. IA genome possesses limited catabolic capacities that, nevertheless, could theoretically support a free-living lifestyle by channeling a narrow range of substrates such as ribose, polyhydroxybutyrate and several amino acids to acetyl-coenzyme A. On the other hand, Cand. IA possesses relatively well-developed anabolic capabilities, although it remains auxotrophic for several amino acids and cofactors. Phylogenetic analysis suggests that the majority of Cand. IA anabolic genes were acquired from bacterial donors via horizontal gene transfer. We thus propose that members of the ‘Diapherotrites'' have evolved from an obligate symbiotic ancestor by acquiring anabolic genes from bacteria that enabled independent biosynthesis of biological molecules previously acquired from symbiotic hosts. ‘Diapherotrites'' 16S rRNA genes exhibit multiple mismatches with the majority of archaeal 16S rRNA primers, a fact that could be responsible for their observed rarity in amplicon-generated data sets. The limited substrate range, complex growth requirements and slow growth rate predicted could be responsible for its refraction to isolation.     

Seasonal and Spatial Variability of Bacterial and Archaeal Assemblages in the Coastal Waters near Anvers Island, Antarctica

A previous report of high levels of members of the domain Archaea in Antarctic coastal waters prompted us to investigate the ecology of Antarctic planktonic prokaryotes. rRNA hybridization techniques and denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial V3 region were used to study variation in Antarctic picoplankton assemblages. In Anvers Island nearshore waters during late winter to early spring, the amounts of archaeal rRNA ranged from 17.1 to 3.6% of the total picoplankton rRNA in 1996 and from 16.0 to 1.0% of the total rRNA in 1995. Offshore in the Palmer Basin, the levels of archaeal rRNA throughout the water column were higher (average, 24% of the total rRNA) during the same period in 1996. The archaeal rRNA levels in nearshore waters followed a highly seasonal pattern and markedly decreased during the austral summer at two stations. There was a significant negative correlation between archaeal rRNA levels and phytoplankton levels (as inferred from chlorophyll a concentrations) in nearshore surface waters during the early spring of 1995 and during an 8-month period in 1996 and 1997. In situ hybridization experiments revealed that 5 to 14% of DAPI (4′,6-diamidino-2-phenylindole)-stained cells were archaeal, corresponding to 0.9 × 10⁴ to 2.7 × 10⁴ archaeal cells per ml, in late winter 1996 samples. Analysis of bacterial ribosomal DNA fragments by DGGE revealed that the assemblage composition may reflect changes in water column stability, depth, or season. The data indicate that changes in Antarctic seasons are accompanied by significant shifts in the species composition of bacterioplankton assemblages and by large decreases in the relative proportion of archaeal rRNA in the nearshore water column.     

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°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.     

Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae

A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15°C and slow growth at 20 and 25°C. Growth of a nop12Δ strain at 15 and 20°C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.     

Stenoxybacter acetivorans gen. nov., sp. nov., an Acetate-Oxidizing Obligate Microaerophile among Diverse O2-Consuming Bacteria from Termite Guts

In termite hindguts, fermentative production of acetate—a major carbon and energy source for the insect—depends on efficient removal of inwardly diffusing oxygen by microbes residing on and near the hindgut wall. However, little is known about the identity of these organisms or about the substrate(s) used to support their respiratory activity. A cultivation-based approach was used to isolate O₂-consuming organisms from hindguts of Reticulitermes flavipes. A consistently greater (albeit not statistically significant) number of colonies developed under hypoxia (2% [vol/vol] O₂) than under air, and the increase coincided with the appearance of morphologically distinct colonies of a novel, rod-shaped, obligately microaerophilic β-proteobacterium that was <95% similar (based on the 16S rRNA gene sequence) to its closest known relative (Eikenella corrodens). Nearly identical organisms (and/or their 16S rRNA genes) were obtained from geographically separated and genetically distinct populations of Reticulitermes. PCR-based procedures implied that the novel isolates were autochthonous to the hindgut of R. flavipes and comprised ca. 2 to 7% of the hindgut prokaryote community. Representative strain TAM-DN1 utilized acetate and a limited range of other organic and amino acids as energy sources and possessed catalase and superoxide dismutase. On solid medium, the optimal O₂ concentration for growth was about 2%, and no growth occurred with O₂ concentrations above 4% or under anoxia. However, cells in liquid medium could grow with higher O₂ concentrations (up to 16%), but only after proportionately extended lag phases. The genetic and physiological distinctiveness of TAM-DN1 and related strains supports their recognition as a new genus and species, for which the name Stenoxybacter acetivorans gen. nov., sp. nov. is proposed.     

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.