Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “Pseudomonas butanovora”

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium “Pseudomonas butanovora.” Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ~20 nmol mg protein⁻¹ of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein⁻¹. Oxidation of similar amounts of each DCE isomer (~20 nmol mg protein⁻¹) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown “P. butanovora” did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.     

Reductive Dechlorination of Chlorinated Ethenes and 1,2-Dichloroethane by “Dehalococcoides ethenogenes” 195

“Dehalococcoides ethenogenes” 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

Site-directed amino acid substitutions in the hydroxylase alpha subunit of butane monooxygenase from Pseudomonas butanovora: Implications for substrates knocking at the gate

Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase alpha subunit of butane monooxygenase (BMOH-alpha) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-alpha were altered to the corresponding residues from the alpha subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 microM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 microM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-alpha.     

Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium "Pseudomonas butanovora"

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of approximately 20 nmol mg protein(-1) of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein(-1). Oxidation of similar amounts of each DCE isomer ( approximately 20 nmol mg protein(-1)) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.     

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

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.     

Isolation and characterization of tetrachloroethylene- and <Emphasis Type="Italic">cis</Emphasis>-1,2-dichloroethylene-dechlorinating propionibacteria

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹⁴C] PCE and [1,2-¹⁴C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.     

Oxidation of Trichloroethylene, 1,1-Dichloroethylene, and Chloroform by Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, which oxidizes toluene and o-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-DCE, trans-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5,6-pentachlorophenol. Escherichia coli JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the lac promoter degraded TCE (3.3 μM), 1,1-DCE (1.25 μM), and chloroform (6.3 μM) at initial rates of 3.1, 3.6, and 1.6 nmol/(min · mg of protein), respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization (2.6, 1.5, and 2.3 Cl⁻ atoms per molecule of TCE, 1,1-DCE, and chloroform, respectively). Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.     

Diversity in Butane Monooxygenases among Butane-Grown Bacteria

Butane monooxygenases of butane-grown Pseudomonas butanovora, Mycobacterium vaccae JOB5, and an environmental isolate, CF8, were compared at the physiological level. The presence of butane monooxygenases in these bacteria was indicated by the following results. (i) O₂ was required for butane degradation. (ii) 1-Butanol was produced during butane degradation. (iii) Acetylene inhibited both butane oxidation and 1-butanol production. The responses to the known monooxygenase inactivator, ethylene, and inhibitor, allyl thiourea (ATU), discriminated butane degradation among the three bacteria. Ethylene irreversibly inactivated butane oxidation by P. butanovora but not by M. vaccae or CF8. In contrast, butane oxidation by only CF8 was strongly inhibited by ATU. In all three strains of butane-grown bacteria, specific polypeptides were labeled in the presence of [¹⁴C]acetylene. The [¹⁴C]acetylene labeling patterns were different among the three bacteria. Exposure of lactate-grown CF8 and P. butanovora and glucose-grown M. vaccae to butane induced butane oxidation activity as well as the specific acetylene-binding polypeptides. Ammonia was oxidized by all three bacteria. P. butanovora oxidized ammonia to hydroxylamine, while CF8 and M. vaccae produced nitrite. All three bacteria oxidized ethylene to ethylene oxide. Methane oxidation was not detected by any of the bacteria. The results indicate the presence of three distinct butane monooxygenases in butane-grown P. butanovora, M. vaccae, and CF8.     

Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes

The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.     

Discrimination of Multiple Dehalococcoides Strains in a Trichloroethene Enrichment by Quantification of Their Reductive Dehalogenase Genes

While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.     

Trichloroethene Reductive Dehalogenase from Dehalococcoides ethenogenes: Sequence of tceA and Substrate Range Characterization

The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoid-containing enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 μmol/min/mg, respectively, similar to the rates for TCE, cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP)₂. Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Sec-independent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.     

Characterization of a newly isolatedcis-1,2-dichloroethylene and aliphatic compound-degrading bacterium,Clostridium sp. strain KYT-1

Acis-1,2-dichloroethylene (cis-DCE)-degrading anaerobic bacterium,Clostridium sp. strain KYT-1, was isolated from a sediment sample collected from a landfill site in Nanji-do, Seoul, Korea. The KYT-1 strain is a gram-positive, endospore-forming, motile, rod-shaped anaerobic bacterium, of approximately 2.5∼3.0 μm in length. The degradation ofcis-DCE is closely related with the growth of the KYT-1 strain, and it was stopped when the growth of the KYT-1 strain became constant. Although the pathway ofcis-DCE degradation by strain KYT-1 remains to be further elucidated, no accumulation of the harmful intermediate, vinyl chloride (VC), was observed during anaerobiccis-DCE degradation. Strain KYT-1 proved able to degrade a variety of volatile organic compounds, including VC, isomers of DCE (1,1-dichloroethylene,trans-1,2-dichloroethylene, andcis-DCE), trichloroethylene, tetrachloroethylene, 1,2-dichloroethane, 1,1,1-trichloroethane, and 1,1,2-trichloroethane. Strain KYT-1 degradedcis-DCE at a range of temperatures from 15 to 37°C, with an optimum at 30°C, and at a pH range of 5.5 to 8.5, with an optimum at 7.0.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.

     

Effects of Chloromethanes on Growth of and Deletion of the pce Gene Cluster in Dehalorespiring Desulfitobacterium hafniense Strain Y51

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 μM, but not carbon dichloride even at 1 mM.     

Tetrachloroethene Dehalorespiration and Growth of Desulfitobacterium frappieri TCE1 in Strict Dependence on the Activity of Desulfovibrio fructosivorans

Tetrachloroethene (PCE) dehalorespiration was investigated in a continuous coculture of the sulfate-reducing bacterium Desulfovibrio fructosivorans and the dehalorespiring Desulfitobacterium frappieri TCE1 at different sulfate concentrations and in the absence of sulfate. Fructose (2.5 mM) was the single electron donor, which could be used only by the sulfate reducer. With 2.5 mM sulfate, the dehalogenating strain was outnumbered by the sulfate-reducing bacterium, sulfate reduction was the dominating process, and only trace amounts of PCE were dehalogenated by strain TCE1. With 1 mM sulfate in the medium, complete sulfate reduction and complete PCE dehalogenation to cis-dichloroethene (cis-DCE) occurred. In the absence of sulfate, PCE was also completely dehalogenated to cis-DCE, and the population size of strain TCE1 increased significantly. The results presented here describe for the first time dehalogenation of PCE by a dehalorespiring anaerobe in strict dependence on the activity of a sulfate-reducing bacterium with a substrate that is exclusively used by the sulfate reducer. This interaction was studied under strictly controlled and quantifiable conditions in continuous culture and shown to depend on interspecies hydrogen transfer under sulfate-depleted conditions. Interspecies hydrogen transfer was demonstrated by direct H₂ measurements of the gas phase and by the production of methane after the addition of a third organism, Methanobacterium formicicum.     

Reductive dechlorination of chlorinated ethenes and 1, 2-dichloroethane by "Dehalococcoides ethenogenes" 195.

"Dehalococcoides ethenogenes" 195 can reductively dechlorinate tetrachloroethene (PCE) completely to ethene (ETH). When PCE-grown strain 195 was transferred (2% [vol/vol] inoculum) into growth medium amended with trichloroethene (TCE), cis-dichloroethene (DCE), 1,1-DCE, or 1,2-dichloroethane (DCA) as an electron acceptor, these chlorinated compounds were consumed at increasing rates over time, which indicated that growth occurred. Moreover, the number of cells increased when TCE, 1,1-DCE, or DCA was present. PCE, TCE, 1,1-DCE, and cis-DCE were converted mainly to vinyl chloride (VC) and then to ETH, while DCA was converted to ca. 99% ETH and 1% VC. cis-DCE was used at lower rates than PCE, TCE, 1,1-DCE, or DCA was used. When PCE-grown cultures were transferred to media containing VC or trans-DCE, products accumulated slowly, and there was no increase in the rate, which indicated that these two compounds did not support growth. When the intermediates in PCE dechlorination by strain 195 were monitored, TCE was detected first, followed by cis-DCE. After a lag, VC, 1,1-DCE, and trans-DCE accumulated, which is consistent with the hypothesis that cis-DCE is the precursor of these compounds. Both cis-DCE and 1,1-DCE were eventually consumed, and both of these compounds could be considered intermediates in PCE dechlorination, whereas the small amount of trans-DCE that was produced persisted. Cultures grown on TCE, 1,1-DCE, or DCA could immediately dechlorinate PCE, which indicated that PCE reductive dehalogenase activity was constitutive when these electron acceptors were used.     

Isolation and Characterization of “Dehalococcoides” sp. Strain MB,Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H₂ as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.Dehalorespiring bacteria play an important role in the transformation and detoxification of a wide range of halogenated compounds, e.g., chlorophenols, chloroethenes, chlorobenzenes, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) (2, 4, 9, 14, 16, 17, 32, 35, 38). Among these compounds, the organic solvents tetrachloroethene (PCE) and trichloroethene (TCE) are suspected carcinogens that are found in soil and groundwater due to their extensive usage and improper disposal (6). The widespread PCE and TCE in the subsurface environment have driven intensive studies of anaerobic microbes capable of reductive dechlorination of chloroethenes (40). Over the last decade, at least 18 isolates, which belong to the genera Desulfitobacterium, Sulfurospirillum, Desulfomonile, Desulfuromonas, Geobacter, “Dehalococcoides,” and Dehalobacter, show reductive dechlorination of chlorinated ethenes (16, 40). In particular, most of these microbes produce cis-1,2-dichloroethene (cis-DCE) as the end product in the chloroethene-contaminated sites, whereas complete detoxification of PCE or TCE to ethene has been restricted only to members of the genus Dehalococcoides. Thus, the Dehalococcoides species have received considerable attention from the bioremediation community in the past decade.Several strains of Dehalococcoides species (e.g., 195, CBDB1, BAV1, and VS) have been sequenced for their whole genomes (24, 39). Their dechlorinating capabilities have also been well addressed through identification and quantification of the known chloroethene reductive dehalogenase (RDase) genes or expression of specific RDase genes (18, 21, 25, 41). In chloroethene-contaminated sites, the natural activities of single or multiple Dehalococcoides strains can lead either to more-toxic, mobile intermediates (e.g., cis- or trans-DCEs and vinyl chloride [VC]) via partial dechlorination of PCE/TCE or to harmless ethene by complete detoxification (10, 13, 15, 41). Many mixed cultures and pure isolates have been reported to produce cis-DCE or VC during PCE/TCE dechlorination processes (15, 40, 43). However, trans-DCE has been detected in more than one-third of the U.S. Environmental Protection Agency (EPA) superfund sites (3a). The source of trans-DCE production was thought to be an abiotic process; however, recently both trans-DCE generation and cis-DCE generation were reported to occur via microbial dechlorination.To date, microbes from either Dehalococcoides- or DF-1-containing mixed cultures have been reported to produce more trans- than cis-DCE, with a ratio of 1.2:1 to 3.5:1 in laboratory-scale studies (8, 10, 22, 31). For example, in a recent report by Kittelmann and Friedrich (22), trans-/cis-DCE at a ratio of 3.5:1 was generated in tidal flat sediment-containing microcosms with microbes closely related to Dehalococcoides sp. or DF-1-like microbes. Additionally, Griffin et al. identified Dehalococcoides species of the Pinellas subgroup in several enrichment cultures, which dechlorinated TCE (∼0.25 mM) to trans-DCE and cis-DCE at a ratio of ∼3:1 (10). There is no information available on the Dehalococcoides isolates that generate trans-DCE as the main end product. This also means a lack of information on the genomic contents of trans-DCE-producing bacteria. Therefore, finding microorganisms that produce trans-DCE in pure culture will be useful for the comprehensive characterization of this group of bacteria.The aim of this study was to isolate a PCE-to-trans-DCE-dechlorinating culture to facilitate the elucidation of trans-DCE formation during reductive dechlorination processes. Microarray analysis was conducted to compare the whole-genome contents of the new isolate and the well-characterized Dehalococcoides ethenogenes strain 195 (30). In addition, a coculture which consisted of the new isolate and TCE-to-cis-DCE-to-VC-dechlorinating Dehalococcoides sp. strain ANAS1 was explored to study the interaction, distribution, and function of the dechlorinators in the dechlorinating process.     

Role of trehalose synthesis pathways in salt tolerance mechanism of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> f. sp. <Emphasis Type="Italic">denitrificans</Emphasis> IL106

Two strains of trichloroethylene (TCE)-degrading bacteria were isolated from soils at polluted and unpolluted sites. The isolates, strains TE26^T and K6, showed co-substrate-independent TCE-degrading activity. TCE degradation was accelerated by preincubation with tetrachloroethylene, cis-dichloroethylene (DCE) and 1,1-DCE. TCE-degrading activities of strains TE26^T and K6 were 0.23, 0.24 mol min^–1 g^–1 dry cells, respectively. 16S rDNA sequences of strains TE26^T and K6 were almost identical (99.7% similarity), and most closely related to Ralstonia basilensis (ATCC17697^T) (98.5% similarity). From the results of DNA–DNA hybridizations, strain TE26^T was genetically coherent to strain K6 (94 and 88% hybridization), and exhibited lower relatedness to R. basilensis (DSM11853^T) (44% and 15%). In addition, because of the differences in chemotaxonomic properties, strain TE26^T and strain K6 appear to be distinct from all established species of the Ralstonia group. Based on these results and the proposal of transferring R. basilensis and related species to Wautersia gen. nov., we propose that these strains should be assigned to the genus Wautersia as Wautersia numadzuensis sp. nov.     

Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.     

Priority pollutant degradation by the facultative methanotroph, Methylocystis strain SB2

Methylocystis strain SB2, a facultative methanotroph capable of growth on multi-carbon compounds, was screened for its ability to degrade the priority pollutants 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA), and 1,1-dichloroethylene (1,1-DCE), as well as cis-dichloroethylene (cis-DCE) when grown on methane or ethanol. Methylocystis strain SB2 degraded 1,2-DCA and 1,1,2-TCA when grown on either substrate and cis-DCE when grown on methane. Growth of Methylocystis strain SB2 on methane was inhibited in the presence of all compounds, while only 1,1-DCE and cis-DCE inhibited growth on ethanol. No degradation of any chlorinated hydrocarbon was observed in ethanol-grown cultures when particulate methane monooxygenase (pMMO) activity was inhibited with the addition of acetylene, indicating that competition for binding to the pMMO between the chlorinated hydrocarbons and methane limited both methanotrophic growth and pollutant degradation when this strain was grown on methane. Characterization of Methylocystis strain SB2 found no evidence of a high-affinity form of pMMO for methane, nor could this strain utilize 1,2-DCA or its putative oxidative products 2-chloroethanol or chloroactetic acid as sole growth substrates, suggesting that this strain lacks appropriate dehydrogenases for the conversion of 1,2-DCA to glyoxylate. As ethanol: (1) can be used as an alternative growth substrate for promoting pollutant degradation by Methylocystis strain SB2 as the pMMO is not required for its growth on ethanol and (2) has been used to enhance the mobility of chlorinated hydrocarbons in situ, it is proposed that ethanol can be used to enhance both pollutant transport and biodegradation by Methylocystis strain SB2.