Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains

The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.     

Stable Isotope Fractionation of Tetrachloroethene during Reductive Dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. Strain PCE-S and Abiotic Reactions with Cyanocobalamin

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B₁₂) was studied. Fractionation was largest during the reaction with cyanocobalamin with αC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (αC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (αC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, αC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.     

Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction

Background

Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references.

Results

In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3?C4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported.

Conclusions

Our findings along with other studies show that HA clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous CA E. faecium isolates. Which of these differences are important for the success of specific E. faecium lineages in the hospital environment remain(s) to be determined.     

Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B12) was studied. Fractionation was largest during the reaction with cyanocobalamin with alphaC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (alphaC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (alphaC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, alphaC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.     

Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase

Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS.     

Preparation of silybin and isosilybin sulfates by sulfotransferase from Desulfitobacterium hafniense

Flavonolignans silybin and isosilybin are major components of silymarin complex isolated from seeds of the milk thistle (Silybum marianum) featuring strong antioxidant and hepatoprotective effects, and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Natural silybin and isosilybin are mixtures of diastereoisomers: silybin/isosilybin A (1a, 1b) and silybin/isosilybin B (2a, 2b). The metabolism of these compounds is supposed to be strongly linked to Phase II of biotransformation and the respective conjugates are rapidly excreted in bile and urine. The aim of this study was to obtain optically pure sulfated metabolites of both silybins and isosilybins. Aryl-sulfate sulfotransferase (EC 2.8.2.22) from Desulfitobacterium hafniense was found to be a highly effective tool for the regiospecific enzymatic synthesis of silybin A-20-O-sulfate, silybin B-20-O-sulfate, isosilybin A-20-O-sulfate and isosilybin B-20-O-sulfate providing nearly quantitative yields and employing cheap p-nitrophenyl sulfate as sulfate donor. The isolated sulfated products will be used as authentic standards in metabolic studies of both silybins and isosilybins.     

Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the delta subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

Anaerobic dehalogenation of hydroxylated polychlorinated biphenyls by Desulfitobacterium dehalogenans

Ten years after reports on the existence of anaerobic dehalogenation of polychlorinated biphenyls (PCBs) in sediment slurries, we report here on the rapid reductive dehalogenation of para-hydroxylated PCBs (HO-PCBs), the excreted main metabolites of PCB in mammals, which can exhibit estrogenic and antiestrogenic activities in humans. The anaerobic bacterium Desulfitobacterium dehalogenans completely dehalogenates all flanking chlorines (chlorines in ortho position to the para-hydroxyl group) from congeners such as 3,3',5, 5'-tetrachloro-4,4'-dihydroxybiphenyl.     

Identification and Characterization of a Novel CprA Reductive Dehalogenase Specific to Highly Chlorinated Phenols from Desulfitobacterium hafniense Strain PCP-1

Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent K_m value for PCP was 46.7 μM at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.Halogenated compounds are generally known as toxic environmental pollutants. Hydrogenolytic reductive dehalogenation, a reaction involving the replacement of one halogen atom with one hydrogen atom, is the predominant mechanism for their transformation in anaerobic environments. This process can sustain microbial growth via electron transport-coupled phosphorylation (10, 26, 31). The majority of the known reductive dehalogenases (RDases) belong to the CprA/PceA family. These are single-polypeptide membrane-associated anaerobic enzymes that are synthesized as preproteins with a cleavable twin arginine translocation (TAT) peptide signal. They contain one corrinoid and two iron-sulfur clusters as cofactors.CprA enzymes catalyzing the reductive dechlorination of chloroaromatics have been purified from Desulfitobacterium hafniense strain DCB-2 (6), Desulfitobacterium dehalogenans (30), Desulfitobacterium chlororespirans strain Co23 (12, 14), Desulfitobacterium sp. strain PCE1 (29), and D. hafniense strain PCP-1 (28) and characterized, and PceA enzymes have been purified from Sulfurospirillum multivorans (22, 23), Desulfitobacterium sp. strain PCE-S (18, 19), D. hafniense strain TCE1 (29), Dehalococcoides ethenogenes 195 (15, 16), Desulfitobacterium sp. strain PCE1 (29), Dehalobacter restrictus (17, 25), Desulfitobacterium sp. strain Y51 (27), and Dehalococcoides sp. strain VS (20) and characterized. However, none of these enzymes showed high dechlorinating activity toward highly chlorinated phenols such as pentachlorophenol (PCP).D. hafniense strain PCP-1 is the only known strict anaerobic bacterium which reductively dechlorinates PCP to 3-chlorophenol (3-CP) and a variety of halogenated aromatic compounds at the ortho, meta, and para positions (2, 7). It dechlorinates PCP at the ortho, ortho, para, and meta positions in the following order: PCP → 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP) → 3,4,5-trichlorophenol (3,4,5-TCP) → 3,5-dichlorophenol (3,5-DCP) → 3-CP (7). Several RDases are thought to operate during this sequence of dechlorinations. Two RDases have already been purified from strain PCP-1. The first one, CrdA, is a membrane-associated enzyme, not related to CprA/PceA-type RDases, that mediates ortho dechlorination of 2,4,6-TCP and several chlorophenols (3). The second enzyme, CprA5, catalyzes the meta and para dechlorination of 3,5-DCP and several chlorophenols (28). Three other putative cprA genes were identified in strain PCP-1 (cprA2, cprA3, and cprA4), which suggests that other RDases with different specificities toward halogenated compounds exist in this strain (8, 31, 32). In this study, we have partially purified and characterized a new CprA-type RDase (CprA3) from strain PCP-1. CprA3 is the first reported RDase with high affinity toward PCP and with high ortho-dechlorinating activity toward PCP and other highly chlorinated phenols.     

Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei DCB-1.

Reductive dehalogenation of chlorophenols has been reported in undefined anaerobic cultures but never before in an anaerobic pure culture. We found that the sulfate-reducing bacterium Desulfomonile tiedjei DCB-1 reductively dehalogenates pentachlorophenol (PCP) and other chlorophenols. The maximum rate of PCP dechlorination observed was 54 mu mol of Cl- h-1 g of protein-1. 3-Chlorobenzoate appeared to serve as a required inducer for PCP dehalogenation; however, neither PCP nor 3-chlorophenol induced dehalogenation. Dehalogenation was catalyzed by living cells, and formate served as a required electron donor. D. tiedjei dehalogenated meta-chlorine substituents of chlorophenols (i.e., PCP was degraded to 2,4,6-trichlorophenol). Generally, more highly chlorinated phenol congeners were more readily dechlorinated, and 3-chlorophenol was not dehalogenated. Growing cultures dehalogenated PCP, but greater than 10 microM PCP (approximately 1.7 mmol g of protein-1) reversibly inhibited growth.     

Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei DCB-1.

Reductive dehalogenation of chlorophenols has been reported in undefined anaerobic cultures but never before in an anaerobic pure culture. We found that the sulfate-reducing bacterium Desulfomonile tiedjei DCB-1 reductively dehalogenates pentachlorophenol (PCP) and other chlorophenols. The maximum rate of PCP dechlorination observed was 54 mu mol of Cl- h-1 g of protein-1. 3-Chlorobenzoate appeared to serve as a required inducer for PCP dehalogenation; however, neither PCP nor 3-chlorophenol induced dehalogenation. Dehalogenation was catalyzed by living cells, and formate served as a required electron donor. D. tiedjei dehalogenated meta-chlorine substituents of chlorophenols (i.e., PCP was degraded to 2,4,6-trichlorophenol). Generally, more highly chlorinated phenol congeners were more readily dechlorinated, and 3-chlorophenol was not dehalogenated. Growing cultures dehalogenated PCP, but greater than 10 microM PCP (approximately 1.7 mmol g of protein-1) reversibly inhibited growth.     

Reductive dehalogenation of carbon tetrachloride by Escherichia coli K-12

The formation of radicals from carbon tetrachloride (CT) is often invoked to explain the product distribution resulting from its transformation. Radicals formed by reduction of CT presumably react with constituents of the surrounding milieu to give the observed product distribution. The patterns of transformation observed in this work were consistent with such a hypothesis. In cultures of Escherichia coli K-12, the pathways and rates of CT transformation were dependent on the electron acceptor condition of the media. Use of oxygen and nitrate as electron acceptors generally prevented CT metabolism. At low oxygen levels (approximately 1%), however, transformation of [14C]CT to 14CO2 and attachment to cell material did occur, in accord with reports of CT fate in mammalian cell cultures. Under fumarate-respiring conditions, [14C]CT was recovered as 14CO2, chloroform, and a nonvolatile fraction. In contrast, fermenting conditions resulted in more chloroform, more cell-bound 14C, and almost no 14CO2. Rates of transformation of CT were faster under fermenting conditions than under fumarate-respiring conditions. Transformation rates also decreased over time, suggesting the gradual exhaustion of transformation activity. This loss was modeled with a simple exponential decay term.     

Reductive dehalogenation of tetrachloroethylene by microorganisms: current knowledge and application strategies

Reductive anaerobic dehalogenation is a useful method for remediation of sites contaminated by chlorinated ethylenes, where hydrogen concentration plays the key role. Under anaerobic conditions, dehalogenating bacteria compete best against methanogenic consortia when the hydrogen level is low; and methanogenic consortia outplay dehalogenating bacteria when the hydrogen level is high. Thus, in an anaerobic mixed culture, efficient use of hydrogen for dehalogenation can be achieved by strategies that maintain hydrogen at a certain low concentration. However, due to the role of acetate, expected dehalogenating results cannot be obtained and unexpected methane formation can be encountered in practice.     

Specificity of reductive dehalogenation of substituted ortho-chlorophenols by Desulfitobacterium dehalogenans JW/IU-DC1.

Resting cells of Desulfitobacterium dehalogenans JW/IU-DC1 growth with pyruvate and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor and inducer of dehalogenation reductively ortho-dehalogenate pentachlorophenol (PCP); tetrachlorophenols (TeCPs); the trichlorophenols 2,3,4-TCP, 2,3,6-TCP, and 2,4,6-TCP; the dichlorophenols 2,3-DCP, 2,4-DCP, and 2,6-DCP; 2,6-dichloro-4-R-phenols (2,6-DCl-4-RPs, where R is -H, -F, -Cl, -NO2, -CO2, or -COOCH3; 2-chloro-4-R-phenols (2-Cl-4-RPs, where R is -H, -F, -Cl, -Br, -NO2, -CO2-, -CH2CO2, or -COOCH3); and the bromophenols 2-BrP, 2,6-DBrP, and 2-Br-4ClP [corrected]. Monochlorophenols, the dichlorophenols 2,5-DCP, 3,4-DCP, and 3,5-DCP, the trichlorophenols 2,3,5-TCP, 2,4,5-TCP, and 3,4,5-TCP, and the fluorinated analog of 3-Cl-4-OHPA, 3-F-4-OHPA ("2-F-4-CH2CO2- P"), are not dehalogenated. A chlorine substituent in position 3 (meta), 4 (para), or 6 (second ortho) of the phenolic moiety facilitates ortho dehalogenation in position 2. Chlorine in the 5 (second meta) position has a negative effect on the dehalogenation rate or even prevents dechlorination in the 2 position. In general, 2,6-DCl-4-RPs are dechlorinated faster than the corresponding 2-Cl-4-RPs with the same substituent R in the 4 position. The highest dechlorination rate, however, was found for dechlorination of 2,3-DCP, with a maximal observed first-order rate constant of 19.4 h-1 g (dry weight) of biomass-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reductive dehalogenation of carbon tetrachloride by Escherichia coli K-12.

The formation of radicals from carbon tetrachloride (CT) is often invoked to explain the product distribution resulting from its transformation. Radicals formed by reduction of CT presumably react with constituents of the surrounding milieu to give the observed product distribution. The patterns of transformation observed in this work were consistent with such a hypothesis. In cultures of Escherichia coli K-12, the pathways and rates of CT transformation were dependent on the electron acceptor condition of the media. Use of oxygen and nitrate as electron acceptors generally prevented CT metabolism. At low oxygen levels (approximately 1%), however, transformation of [14C]CT to 14CO2 and attachment to cell material did occur, in accord with reports of CT fate in mammalian cell cultures. Under fumarate-respiring conditions, [14C]CT was recovered as 14CO2, chloroform, and a nonvolatile fraction. In contrast, fermenting conditions resulted in more chloroform, more cell-bound 14C, and almost no 14CO2. Rates of transformation of CT were faster under fermenting conditions than under fumarate-respiring conditions. Transformation rates also decreased over time, suggesting the gradual exhaustion of transformation activity. This loss was modeled with a simple exponential decay term.     

Occurrence of several genes encoding putative reductive dehalogenases in Desulfitobacterium hafniense/frappieri and Dehalococcoides ethenogenes

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].     

Reductive dehalogenation of dichloroanilines by anaerobic microorganisms in fresh and dichlorophenol-acclimated pond sediment

We investigated the transformation of 2,4-dichloroaniline (2,4-DiCA) and 3,4-DiCA to monochloroanilines (CA) in anaerobic pond sediment. Dechlorination of 3,4-DiCA to 3-CA started after a lag period of 3 weeks and was complete after an additional 5 weeks. Although 2,4-DiCA disappeared over 8 weeks, the appearance of a CA product could not be detected. In contrast, anaerobic bacteria in pond sediment acclimated to dehalogenate 2,4-dichlorophenol (2,4-DiCP) or 3,4-DiCP rapidly dechlorinated 2,4-DiCA and 3,4-DiCA without any lag time. By comparison, anaerobic sediment bacteria acclimated to 3,4-DiCA rapidly degraded 3,4-DiCP without a lag. In all cases, the CA products were stable for the duration of the experiments. It is concluded that cross-acclimation occurred.     

Reductive dehalogenation of halocarboxylic acids by the phototrophic genera Rhodospirillum and Rhodopseudomonas.

Type strains of the purple nonsulfur species Rhodospirillum rubrum, Rhodospirillum photometricum, and Rhodopseudomonas palustris grew phototrophically on a number of two- and three-carbon halocarboxylic acids in the presence of CO2, by reductive dehalogenation and assimilation of the resulting acid. Strains of each of these species were able to grow on chloroacetic, 2-bromopropionic, 2-chloropropionic, and 3-chloropropionic acids at a concentration of 2 mM. Only R. palustris DSM 123 was able to grow on bromoacetic acid and then only at a reduced concentration of 1 mM. R. palustris ATCC 33872 (formerly R. rutila) was unable to grow on any of the substrates tested. The ability of these organisms to utilize halocarboxylic acids indicates that they may have a significant role to play in the removal of these environmental pollutants from illuminated anaerobic habitats such as lakes, waste lagoons, sediments of ditches and ponds, mud, and moist soil.