Youlboong Sung

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

ABSTRACT The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.

Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

ABSTRACT A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter−1 day−1, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.

Geobacter lovleyi sp. nov. Strain SZ, a Novel Metal-Reducing and Tetrachloroethene-Dechlorinating Bacterium†

ABSTRACT A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min−1 mg of protein−1, respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H2 was consumed to threshold concentrations of 0.08 ± 0.03 nM, 0.16 ± 0.07 nM, and 0.5 ± 0.06 nM, respectively, and acetate was consumed to 3.0 ± 2.1 nM, 1.2 ± 0.5 nM, and 3.6 ± 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electron-accepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.

Characterization of Two Tetrachloroethene-Reducing, Acetate-Oxidizing Anaerobic Bacteria and Their Description as Desulfuromonas michiganensis sp. nov.

ABSTRACT Two tetrachlorethene (PCE)-dechlorinating populations, designated strains BB1 and BRS1, were isolated from pristine river sediment and chloroethene-contaminated aquifer material, respectively. PCE-to-cis-1,2-dichloroethene-dechlorinating activity could be transferred in defined basal salts medium with acetate as the electron donor and PCE as the electron acceptor. Taxonomic analysis based on 16S rRNA gene sequencing placed both isolates within the Desulfuromonas cluster in the δ subdivision of the Proteobacteria. PCE was dechlorinated at rates of at least 139 nmol min−1 mg of protein−1 at pH values between 7.0 and 7.5 and temperatures between 25 and 30°C. Dechlorination also occurred at 10°C. The electron donors that supported dechlorination included acetate, lactate, pyruvate, succinate, malate, and fumarate but not hydrogen, formate, ethanol, propionate, or sulfide. Growth occurred with malate or fumarate alone, whereas oxidation of the other electron donors depended strictly on the presence of fumarate, malate, ferric iron, sulfur, PCE, or TCE as an electron acceptor. Nitrate, sulfate, sulfite, thiosulfate, and other chlorinated compounds were not used as electron acceptors. Sulfite had a strong inhibitory effect on growth and dechlorination. Alternate electron acceptors (e.g., fumarate or ferric iron) did not inhibit PCE dechlorination and were consumed concomitantly. The putative fumarate, PCE, and ferric iron reductases were induced by their respective substrates and were not constitutively present. Sulfide was required for growth. Both strains tolerated high concentrations of PCE, and dechlorination occurred in the presence of free-phase PCE (dense non-aqueous-phase liquids). Repeated growth with acetate and fumarate as substrates yielded a BB1 variant that had lost the ability to dechlorinate PCE. Due to the 16S rRNA gene sequence differences with the closest relatives and the unique phenotypic characteristics, we propose that the new isolates are members of a new species, Desulfuromonas michiganensis, within the Desulfuromonas cluster of the Geobacteraceae.

Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil

ABSTRACT Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with φ29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

Arthrobacter soli sp. nov., a novel bacterium isolated from wastewater reservoir sediment

A novel Gram-positive bacterium, designated SYB2T, was isolated from wastewater reservoir sediment, and a polyphasic taxonomic study was conducted based on its morphological, physiological, and biochemical features, as well as the analysis of its 16S rRNA gene sequence. During the phylogenetic analysis of the strain SYB2T, results of a 16S rRNA gene sequence analysis placed this bacterium in the genus Arthrobacter within the family Micrococcaceae. SYB2T and Arthrobacter protophormiae ATCC 19271T, the most closely related species, both exhibited a 16S rRNA gene sequence similarity of 98.99%. The genomic DNA G+C content of the novel strain was found to be 62.0 mol%. The predominant fatty acid composition was ante-iso-C15:0, anteiso-C17:0, iso-C16:0, and iso-C15:0. Analysis of 16S rRNA gene sequences and DNA-DNA related-ness, as well as physiological and biochemical tests, showed genotypic and phenotypic differences between strain SYB2T and other Arthrobacter species. The type strain of the novel species was identified as SYB2T (= KCTC 19291T= DSM 19449T).

Phospholipid furan fatty acids and ubiquinone-8 : Lipid biomarkers that may protect Dehalococcoides strains from free radicals

ABSTRACT Dehalococcoides species have a highly restricted lifestyle and are only known to derive energy from reductive dehalogenation reactions. The lipid fraction of two Dehalococcoides isolates, strains BAV1 and FL2, and a tetrachloroethene-to-ethene-dechlorinating Dehalococcoides-containing consortium were analyzed for neutral lipids and phospholipid fatty acids. Unusual phospholipid modifications, including the replacement of unsaturated fatty acids with furan fatty acids, were detected in both Dehalococcoides isolates and the mixed culture. The following three furan fatty acids are reported as present in bacterial phospholipids for the first time: 9-(5-pentyl-2-furyl)-nonanoate (Fu18:2ω6), 9-(5-butyl-2-furyl)-nonanoate (Fu17:2ω5), and 8-(5-pentyl-2-furyl)-octanoate (Fu17:2ω6). The neutral lipids of the Dehalococcoides cultures contained unusually large amounts of benzoquinones (i.e., ubiquinones [UQ]), which is unusual for anaerobes. In particular, the UQ-8 content of Dehalococcoides was 5- to 20-fold greater than that generated in aerobically grown Escherichia coli cultures relative to the phospholipid fatty acid content. Naphthoquinone isoprenologues (MK), which are often found in anaerobically grown bacteria and archaea, were also detected. Dehalococcoides shows a difference in isoprenologue pattern between UQ-8 and MK-5 that is atypical of other bacteria capable of producing both quinone types. The difference in UQ-8 and MK-5 isoprenologue patterns strongly suggests a special function for UQ in Dehalococcoides, and Dehalococcoides may utilize structural modifications in its lipid armamentarium to protect against free radicals that are generated in the process of reductive dechlorination.

Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites

ABSTRACT Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 × 106 and 1 × 104 16S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 × 107 ± 0.1 × 107 per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.

Haloterrigena jeotgali sp. nov., an extremely halophilic archaeon from salt-fermented food.

A novel red-pigmented halophilic archaeon, strain A29T, was isolated from shrimp jeotgal, a traditional salt-fermented food from Korea. This strain grows in the ranges 10-30% (w/v) NaCl, 17-50 degrees C and pH 6.5-8.5, with optimal growth occurring at 15-20% NaCl, 37-45 degrees C and pH 7.0-7.5. The isolate is Gram-negative and non-motile. Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strain A29T is associated with the genus Haloterrigena and closely related to the species Haloterrigena thermotolerans (99.0% similarity). However, DNA-DNA hybridization experiments revealed that the level of hybridization between strain A29T and related strains of Haloterrigena is less than 70%. The polar lipid fraction consists of phosphatidylglyerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and mannose-2,6-disulfate(1-2)-glucose glycerol diether (S2-DGD). The G+C content of genomic DNA of the type strain is 62.3 mol%. On the basis of this polyphasic taxonomic study, strain A29T should be placed in the genus Haloterrigena as a novel species, for which the name Haloterrigena jeotgali sp. nov. is proposed. The type strain of the new species is A29T (=KCTC 4020T=DSM 18794T=JCM 14585T=CECT 7218T).

Characterization of the depth-related changes in the microbial communities in Lake Hovsgol sediment by 16S rRNA gene-based approaches.

The undisturbed sediment of Lake Hovsgol (Mongolia) is scientifically important because it represents a record of the environmental changes that took place between the Holocene (the present age) and Pleistocene (the last ice age; 12,000 14C years before present day). Here, we investigated how the current microbial communities change as the depth increases by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes of the microbial communities. The microbial diversity, as estimated by the Shannon index, decreased as the depth increased. In particular, significant changes in archaeal diversity were observed in the middle depth (at 39–42 cm depth of total 60 cm depth) that marks the border between the Holocene and Pleistocene. Phylotype belonging to Beta-and Gamma-Proteobacteria were the predominant bacteria and most of these persisted throughout the depth examined. However, as the depth increased, some bacteria (some genera belonging to Beta-Proteobacteria, Nitrospira, and OP8-9) were not detectable while others (some genera belonging to Alpha-, Beta-, Gamma-Proteobacteria) newly detected by DGGE. Crenarchaea were the predominant archaea and only one phylotype belonging to Euryarchaea was found. Both the archaeal and bacterial profiles revealed by the DGGE band patterns could be grouped into four and three subsets, respectively, subsets that were largely divided by the border between the Holocene and Pleistocene. Thus, the diversity of the current microbial communities in Lake Hovsgol sediments decreases with increasing depth. These changes probably relate to the environmental conditions in the sediments, which were shaped by the paleoclimatic events taking place between the Holocene and Pleistocene.