Kimberly S. Bowman

Complete genome sequence of Dehalogenimonas lykanthroporepellens type strain (BL-DC-9T) and comparison to “Dehalococcoides” strains

Dehalogenimonas lykanthroporepellens is the type species of the genus Dehalogenimonas, which belongs to a deeply branching lineage within the phylum Chloroflexi. This strictly anaerobic, mesophilic, non spore-forming, Gram-negative staining bacterium was first isolated from chlorinated solvent contaminated groundwater at a Superfund site located near Baton Rouge, Louisiana, USA. D. lykanthroporepellens was of interest for genome sequencing for two reasons: (a) an unusual ability to couple growth with reductive dechlorination of environmentally important polychlorinated aliphatic alkanes and (b) a phylogenetic position that is distant from previously sequenced bacteria. The 1,686,510 bp circular chromosome of strain BL-DC-9T contains 1,720 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small subunit rRNA (16S) locus.

Dehalogenimonas alkenigignens sp. nov., a chlorinated-alkane-dehalogenating bacterium isolated from groundwater.

Two strictly anaerobic bacterial strains, designated IP3-3(T) and SBP-1, were isolated from groundwater contaminated by chlorinated alkanes and alkenes at a Superfund Site located near Baton Rouge, Louisiana (USA). Both strains reductively dehalogenate a variety of polychlorinated aliphatic alkanes, including 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane and 1,2,3-trichloropropane, when provided with hydrogen as the electron donor. To clarify their taxonomic position, strains IP3-3(T) and SBP-1 were characterized using a polyphasic approach. Both IP3-3(T) and SBP-1 are mesophilic, non-spore-forming, non-motile and Gram-stain-negative. Cells of both strains are irregular cocci with diameters of 0.4-1.1 µm. Both are resistant to ampicillin and vancomycin. The genomic DNA G+C contents of strains IP3-3(T) and SBP-1 are 55.5±0.4 and 56.2±0.2 mol% (HPLC), respectively. Major cellular fatty acids include C18 : 1ω9c, C16 : 0, C14 : 0 and C16 : 1ω9c. 16S rRNA gene sequence based phylogenetic analyses indicated that the strains cluster within the phylum Chloroflexi most closely related to but distinct from the species Dehalogenimonas lykanthroporepellens (96.2 % pairwise similarity) and Dehalococcoides mccartyi (90.6 % pairwise similarity). Physiological and chemotaxonomic traits as well as phylogenetic analysis support the conclusion that these strains represent a novel species within the genus Dehalogenimonas for which the name Dehalogenimonas alkenigignens sp. nov. is proposed. The type strain is IP3-3(T) ( = JCM 17062(T) = NRRL B-59545(T)).

Dehalogenimonas spp. can Reductively Dehalogenate High Concentrations of 1,2-Dichloroethane, 1,2-Dichloropropane, and 1,1,2-Trichloroethane.

The contaminant concentrations over which type strains of the species Dehalogenimonas alkenigignens and Dehalogenimonas lykanthroporepellens were able to reductively dechlorinate 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were evaluated. Although initially isolated from an environment with much lower halogenated solvent concentrations, D. alkenigignens IP3-3T was found to reductively dehalogenate chlorinated alkanes at concentrations comparable to D. lykanthroporepellens BL-DC-9T. Both species dechlorinated 1,2-DCA, 1,2-DCP, and 1,1,2-TCA present at initial concentrations at least as high as 8.7, 4.0, and 3.5 mM, respectively. The ability of Dehalogenimonas spp. to carry out anaerobic reductive dechlorination even in the presence of high concentrations of chlorinated aliphatic alkanes has important implications for remediation of contaminated soil and groundwater.

Production of hydrogen by Clostridium species in the presence of chlorinated solvents

Although anaerobic bioremediation of chlorinated organic contaminants in the environment often requires exogenous supply of hydrogen as an electron donor, little is known about the ability of hydrogen-producing bacteria to grow in the presence of chlorinated solvents. In this study, 18 Clostridium strains including nine uncharacterized isolates originating from chlorinated solvent contaminated groundwater were tested to determine their ability to fermentatively produce hydrogen in the presence of three common chlorinated aliphatic groundwater contaminants: 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. Some strains produced hydrogen in media containing concentrations as high as 29.7 mM DCA, 9.8 mM TCA, and 1.1 mM PCE. None of the strains biotransformed chlorinated solvents under the conditions tested. Results demonstrate that many Clostridium species are chlorinated solvent tolerant, producing hydrogen even in the presence of high concentrations of DCA, TCA, and PCE. These findings have important implications for bioremediation of contaminated soil and groundwater.

Pelosinus defluvii sp. nov., isolated from chlorinated solvent-contaminated groundwater, emended description of the genus Pelosinus and transfer of Sporotalea propionica to Pelosinus propionicus comb. nov.

Two anaerobic bacterial strains, designated SHI-1(T) and SHI-2, were isolated from chlorinated solvent-contaminated groundwater. They were found to be identical in phenotypic properties and shared high (98.5-99.8 %) pairwise 16S rRNA gene sequence similarity. Multiple 16S rRNA genes were found to be present in the isolates as well as Pelosinus fermentans DSM 17108(T) and Sporotalea propionica DSM 13327(T). Strains SHI-1(T) and SHI-2 could be differentiated from their closest phylogenetic relatives, P. fermentans DSM 17108(T) and S. propionica DSM 13327(T), on the basis of their phenotypic and phylogenetic properties. The isolates were Gram-negative, spore-forming, motile rods with peritrichous flagella. Growth occurred at 10-42 °C and pH 5.5-8.5. Fermentative growth was observed on Casamino acids, fructose, fumarate, glucose, glycerol, pyruvate and yeast extract. The major organic acids produced from glucose and glycerol fermentation were propionate and acetate. The major organic acids produced from fermentation of fumarate were propionate, acetate and succinate. The major cellular fatty acids were summed feature 4 (consisting of C(15:1)ω8c and/or C(15:2)), summed feature 8 (consisting of C(17:1)ω8c and/or C(17:2)) and C(14:0) dimethyl aldehyde. The polar lipids comprised aminophospholipids, including phosphatidylethanolamine and phosphatidylserine, and an unknown phospholipid. The genomic DNA G+C content was 39.2 mol%. We propose that strains SHI-1(T) and SHI-2 are assigned to a novel species of the genus Pelosinus, with the name Pelosinus defluvii sp. nov. (type strain SHI-1(T) = NRRL Y-59407(T) = LMG 25549(T)). The description of the genus Pelosinus is emended. We also propose the transfer of S. propionica to the genus Pelosinus as Pelosinus propionicus comb. nov. (type strain TmPN3(T) = DSM 13327(T) = ATCC BAA-626(T)), on the basis of phylogenetic, chemotaxonomic and phenotypic properties.

Clostridium hydrogeniformans sp. nov. and Clostridium cavendishii sp. nov., hydrogen-producing bacteria from chlorinated solvent-contaminated groundwater

Four hydrogen-producing, aerotolerant, anaerobic bacterial strains isolated from chlorinated solvent-contaminated groundwater were characterized using a polyphasic approach. Three of the strains, designated BL-18, BL-19 and BL-20(T), were found to be identical in 16S rRNA gene sequences and in phenotypic properties. Cells of these strains are Gram-positive-staining, spore-forming, motile rods with peritrichous flagella. Growth occurred at 15-40 degrees C, pH 5.0-10.0 and at NaCl concentrations up to 5 % (w/v). Acid was produced in fermentation of cellobiose, fructose, galactose (weak), glucose, maltose and salicin. Products of fermentation in PYG medium were acetate, butyrate, ethanol, formate, carbon dioxide and hydrogen. Dominant cellular fatty acids when grown in PYG medium were C(13 : 0) iso, C(16 : 0), C(13 : 0) anteiso, C(15 : 0) iso and C(15 : 0) anteiso. The genomic DNA G+C content was 30.4 mol%. These isolates can be differentiated from their closest phylogenetic relative, the cluster I Clostridium species Clostridium frigidicarnis (97.2 % similar to the type strain in 16S rRNA gene sequence), on the basis of phenotypic and chemotaxonomic properties. The other strain characterized in this study, BL-28(T), was Gram-positive-staining with spore-forming, rod-shaped cells. Growth occurred at 15-46 degrees C, pH 6.0-8.5 and at NaCl concentrations up to 3 % (w/v). Acid was produced from cellobiose, dextran, fructose (weak), glucose, maltose, salicin and trehalose. End products of PYG fermentation included acetate, butyrate, pyruvate, carbon dioxide and hydrogen. Dominant cellular fatty acids from cells grown in PYG medium at 30 degrees C were C(14 : 0), C(14 : 0) dimethyl aldehyde, C(16 : 0) and C(12 : 0). The DNA G+C content was 28.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BL-28(T) falls within cluster I of the genus Clostridium, but with <or=95.2 % identity with previously described species. On the basis of results presented here, strains BL-20(T) (=NRRL B-51348(T) =DSM 21757(T)) and BL-28(T) (=NRRL B-51352(T) =DSM 21758(T)) are proposed as the type strains of novel species of the genus Clostridium with the names Clostridium hydrogeniformans sp. nov. and Clostridium cavendishii sp. nov., respectively.

Reassessment of PCR primers targeting 16S rRNA genes of the organohalide-respiring genus Dehalogenimonas

Representatives from the genus Dehalogenimonas have the metabolic capacity to anaerobically transform a variety of environmentally important polychlorinated aliphatic compounds. In light of the recent isolation of additional strains, description of a new species, and an expanded number of uncultured DNA sequences, PCR primers and protocols intended to uniquely target members of this organohalide-respiring genus were reevaluated. Nine of fourteen primer combinations reported previously as genus-specific failed to amplify 16S rRNA genes of recently isolated Dehalogenimonas strains. Use of alternative combinations or modified genus-specific primers, however, allowed detection of all presently known Dehalogenimonas strains. Use of a modified primer set in qPCR revealed an approximately two-order of magnitude increase in concentration of Dehalogenimonas 16S rRNA gene copies following subsurface injection of electron donors at a Louisiana Superfund site, demonstrating the utility of the newly developed protocol and suggesting that the genus Dehalogenimonas can respond to biostimulation remediation strategies in a manner similar to that previously reported for other dechlorinating genera such as Dehalococcoides.

Dehalogenimonas lykanthroporepellens BL-DC-9T simultaneously transcribes many rdhA genes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors.

The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression.

Substrate interactions in dehalogenation of 1,2-dichloroethane, 1,2-dichloropropane, and 1,1,2-trichloroethane mixtures by Dehalogenimonas spp.

When chlorinated alkanes are present as soil or groundwater pollutants, they often occur in mixtures. This study evaluated substrate interactions during the anaerobic reductive dehalogenation of chlorinated alkanes by the type strains of two Dehalogenimonas species, D. lykanthroporepellens and D.alkenigignens. Four contaminant mixtures comprised of combinations of the chlorinated solvents 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were assessed for each species. Chlorinated solvent depletion and daughter product formation determined as a function of time following inoculation into anaerobic media revealed preferential dechlorination of 1,1,2-TCA over both 1,2-DCA and 1,2-DCP for both species. 1,2-DCA in particular was not dechlorinated until 1,1,2-TCA reached low concentrations. In contrast, both species concurrently dechlorinated 1,2-DCA and 1,2-DCP over a comparably large concentration range. This is the first report of substrate interactions during chlorinated alkane dehalogenation by pure cultures, and the results provide insights into the chlorinated alkane transformation processes that may be expected for contaminant mixtures in environments where Dehalogenimonas spp. are present.

Dehalogenimonas formicexedens sp. nov., a chlorinated alkane respiring bacterium isolated from contaminated groundwater.

A strictly anaerobic, Gram-stain-negative, non-spore-forming bacterium designated NSZ-14T, isolated from contaminated groundwater in Louisiana (USA), was characterized using a polyphasic approach. Strain NSZ-14T reductively dehalogenated a variety of polychlorinated aliphatic alkanes, producing ethene from 1,2-dichloroethane, propene from 1,2-dichloropropane, a mixture of cis- and trans-1,2-dichloroethene from 1,1,2,2-tetrachloroethane, vinyl chloride from 1,1,2-trichloroethane and allyl chloride (3-chloro-1-propene) from 1,2,3-trichloropropane. Formate or hydrogen could both serve as electron donors. Dechlorination occurred between pH 5.5 and 7.5 and over a temperature range of 20-37 °C. Major cellular fatty acids included C18 : 1ω9c, C14 : 0 and C16 : 0. 16S rRNA gene sequence-based phylogenetic analysis indicated that the strain clusters within the class Dehalococcoidia of the phylum Chloroflexi, most closely related to but distinct from type strains of the species Dehalogenimonas alkenigignens (97.63 % similarity) and Dehalogenimonas lykanthroporepellens (95.05 %). A complete genome sequence determined for strain NSZ-14T revealed a DNA G+C content of 53.96 mol%, which was corroborated by HPLC (54.1±0.2 mol% G+C). Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strain NSZ-14T represents a novel species within the genus Dehalogenimonas, for which the name Dehalogenimonas formicexedens sp. nov. is proposed. The type strain is NSZ-14T (=HAMBI 3672T=JCM 19277T=VKM B-3058T). An emended description of Dehalogenimonas alkenigignens is also provided.