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.     

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.     

Characterization of four TCE-dechlorinating microbial enrichments grown with different cobalamin stress and methanogenic conditions

To investigate the important supportive microorganisms responsible for trichloroethene (TCE) bioremediation under specific environmental conditions and their relationship with Dehalococcoides (Dhc), four stable and robust enrichment cultures were generated using contaminated groundwater. Enrichments were maintained under four different conditions exploring two parameters: high and low TCE amendments (resulting in inhibited and uninhibited methanogenic activity, respectively) and with and without vitamin B₁₂ amendment. Lactate was supplied as the electron donor. All enrichments were capable of reductively dechlorinating TCE to vinyl chloride and ethene. The dechlorination rate and ethene generation were higher, and the proportion of electrons used for dechlorination increased when methanogenesis was inhibited. Biologically significant cobalamin biosynthesis was detected in the enrichments without B₁₂ amendment. Comparative genomics using a genus-wide microarray revealed a Dhc genome similar to that of strain 195 in all enrichments, a strain that lacks the major upstream corrin ring biosynthesis pathway. Seven other bacterial operational taxonomic units (OTUs) were detected using clone libraries. OTUs closest to Pelosinus, Dendrosporobacter, and Sporotalea (PDS) were most dominant. The Clostridium-like OTU was most affected by B₁₂ amendment and active methanogenesis. Principal component analysis revealed that active methanogenesis, rather than vitamin B₁₂ limitation, exerted a greater effect on the community structures even though methanogens did not seem to play an essential role in providing corrinoids to Dhc. In contrast, acetogenic bacteria that were abundant in the enrichments, such as PDS and Clostridium sp., may be potential corrinoid providers for Dhc.     

Comparative genomics of "Dehalococcoides ethenogenes" 195 and an enrichment culture containing unsequenced "Dehalococcoides" strains

Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by some "Dehalococcoides" organisms. A Dehalococcoides-organism-containing microbial consortium (referred to as ANAS) with the ability to degrade TCE to ethene, an innocuous end product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of "Dehalococcoides ethenogenes" 195. This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray results revealed that the genes associated with central metabolism, including an apparently incomplete carbon fixation pathway, cobalamin-salvaging system, nitrogen fixation pathway, and five hydrogenase complexes, are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase, tceA, was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria, which is essential in developing effective strategies for the bioremediation of PCE and TCE in the environment.     

Anaerobic Bioremediation of Groundwater Containing a Mixture of 1,1,2,2-Tetrachloroethane and Chloroethenes

This study investigated the biotransformation pathways of 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA) in the presence of chloroethenes (i.e. tetrachloroethene, PCE; trichloroethene, TCE) in anaerobic microcosms constructed with subsurface soil and groundwater from a contaminated site. When amended with yeast extract, lactate, butyrate, or H₂ and acetate, 1,1,2,2-TeCA was initially dechlorinated via both hydrogenolysis to 1,1,2-trichloroethane (1,1,2-TCA) (major pathway) and dichloroelimination to dichloroethenes (DCEs) (minor pathway), with both reactions occurring under sulfidogenic conditions. In the presence of only H₂, the hydrogenolysis of 1,1,2,2-TeCA to 1,1,2-TCA apparently required the presence of acetate to occur. Once formed, 1,1,2-TCA was degraded predominantly via dichloroelimination to vinyl chloride (VC). Ultimately, chloroethanes were converted to chloroethenes (mainly VC and DCEs) which persisted in the microcosms for very long periods along with PCE and TCE originally present in the groundwater. Hydrogenolysis of chloroethenes occurred only after highly reducing methanogenic conditions were established. However, substantial conversion to ethene (ETH) was observed only in microcosms amended with yeast extract (200 mg/l), suggesting that groundwater lacked some nutritional factors which were likely provided to dechlorinating microorganisms by this complex organic substrate. Bioaugmentation with an H₂-utilizing PCE-dechlorinating Dehalococcoides spp. -containing culture resulted in the conversion of 1,1,2,2-TeCA, PCE and TCE to ETH and VC. No chloroethanes accumulated during degradation suggesting that 1,1,2,2-TeCA was degraded through initial dichloroelimination into DCEs and then typical hydrogenolysis into ETH and VC.     

Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a Dehalococcoides Species

A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 μmol liter⁻¹day⁻¹, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (K_S) for VC was 5.8 μM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30°C, and negligible dechlorination occurred at 4 and 35°C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H₂ as electron donor. VC-dechlorinating cultures consumed H₂ to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.     

Comparative genomics of two newly isolated Dehalococcoides strains and an enrichment using a genus microarray

Comparative genomics of Dehalococcoides strains and an enrichment were performed using a microarray targeting genes from all available sequenced genomes of the Dehalococcoides genus. The microarray was designed with 4305 probe sets to target 98.6% of the open-reading frames from strains 195, CBDB1, BAV1 and VS. The microarrays were validated and applied to query the genomes of two recently isolated Dehalococcoides strains, ANAS1 and ANAS2, and their enrichment source (ANAS) to understand the genome–physiology relationships. Strains ANAS1 and ANAS2 can both couple the reduction of trichloroethene, cis-dichloroethene (DCE) and 1,1-DCE, but not tetrachloroethene and trans-DCE with growth, whereas only strain ANAS2 couples vinyl chloride reduction to growth. Comparative genomic analysis showed that the genomes of both strains are similar to each other and to strain 195, except for genes that are within the previously defined integrated elements or high-plasticity regions. Combined results of the two isolates closely matched the results obtained using genomic DNA of the ANAS enrichment. The genome similarities, together with the distinct chlorinated ethene usage of strains ANAS1, ANAS2 and 195 demonstrate that closely phylogenetically related strains can be physiologically different. This incongruence between physiology and core genome phylogeny seems to be related to the presence of distinct reductive dehalogenase-encoding genes with assigned chlorinated ethene functions (pceA, tceA in strain 195; tceA in strain ANAS1; vcrA in strain ANAS2). Overall, the microarrays are a valuable high-throughput tool for comparative genomics of unsequenced Dehalococcoides-containing samples to provide insights into their gene content and dechlorination functions.     

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.

     

Aerobic Mineralization of Trichloroethylene, Vinyl Chloride, and Aromatic Compounds by Rhodococcus Species

Two Rhodococcus strains which were isolated from a trichloroethylene (TCE)-degrading bacterial mixture and Rhodococcus rhodochrous ATCC 21197 mineralized vinyl chloride (VC) and TCE. Greater than 99.9% of a 1-mg/liter concentration of VC was degraded by cell suspensions. [1,2-¹⁴C]VC was degraded by cell suspensions, with the production of greater than 66% ¹⁴CO₂ and 20% ¹⁴C-aqueous phase products and incorporation of 10% of the ¹⁴C into the biomass. Cultures that utilized propane as a substrate were able to mineralize greater than 28% of [1,2-¹⁴C]TCE to ¹⁴CO₂, with approximately 40% appearing in ¹⁴C-aqueous phase products and another 10% of ¹⁴C incorporated into the biomass. VC degradation was oxygen dependent and occurred at a pH range of 5 to 10 and temperatures of 4 to 35°C. Cell suspensions degraded up to 5 mg of TCE per liter and up to 40 mg of VC per liter. Propane competitively inhibited TCE degradation. Resting cell suspensions also degraded other chlorinated aliphatic hydrocarbons, such as chloroform, 1,1-dichloroethylene, and 1,1,1-trichloroethane. The isolates degraded a mixture of aromatic and chlorinated aliphatic solvents and utilized benzene, toluene, sodium benzoate, naphthalene, biphenyl, and n-alkanes ranging in size from propane to hexadecane as carbon and energy sources. The environmental isolates appeared more catabolically versatile than R. rhodochrous ATCC 21197. The data report that environmental isolates of Rhodococcus species and R. rhodochrous ATCC 21197 have the potential to degrade TCE and VC in addition to a variety of aromatic and chlorinated aliphatic compounds either individually or in mixtures.     

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.     

Synthetic S-(2,3-dihydroxypropyl)-cysteinyl peptides derived from the N-terminus of the cytochrome subunit of the photoreaction centre of Rhodopseudomonas viridis enhance murine splenocyte proliferation

Various lipopeptides representing the N-terminal part of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas virdis were prepared by solid-phase peptide synthesis. These lipopeptides consisted of a S-[2,3-dihydroxypropyl]-cysteinyl (Dhc) residue N-terminally coupled to the nonapeptide FEPPPATTT. Different numbers of palmitoyl (Pam) chains were attached to Dhc via ester and/or amide bonds. The lipopeptide Dhc(Pam)₂-FEPPPATTT containing two ester-bonded palmitoyl residues and a free N-chain lipopeptide Pam-Dhc(Pam)-FEPPPATTT containing one amide- and one ester-bonded palmitoyl residues, the two-chain lipopeptide Pam-Dhc(Pam)-FEPPPATTT containing one amide- and one ester-bonded palmitoyl residue, and the N-terminally elongated lipopeptide SLVAG-Dhc(Pam)₂-FEPPPATTT were less active. The nonapeptide FEPPPATTT and the decapeptide Dhc-FEPPPATTT were only merginal splenocyte activators, even at concentrations as high as 1 μM . Thus, lipopeptide Dhc(Pam)₂-FEPPPATTT constitutes the first potent splenocyte stimulating Dhc-lipopeptide described so far that contains only two fatty acid residues.     

Analysis of Nicotiana tabacum PIN genes identifies NtPIN4 as a key regulator of axillary bud growth

The plant‐specific PIN‐FORMED (PIN) auxin efflux proteins have been well characterized in many plant species, where they are crucial in the regulation of auxin transport in various aspects of plant development. However, little is known about the exact roles of the PIN genes during plant development in Nicotiana species. This study investigated the PIN genes in tobacco (Nicotiana tabacum) and in two ancestral species (Nicotiana sylvestris and Nicotiana tomentosiformis). Genome‐wide analysis of the N. tabacum genome identified 20 genes of the PIN family. An in‐depth phylogenetic analysis of the PIN genes of N. tabacum, N. sylvestris and N. tomentosiformis was conducted. NtPIN4 expression was strongly induced by the application of exogenous indole‐3‐acetic acid (IAA), but was downregulated by the application of ABA, a strigolactone analogue, and cytokinin, as well as by decapitation treatments, suggesting that the NtPIN4 expression level is likely positively regulated by auxin. Expression analysis indicated that NtPIN4 was highly expressed in tobacco stems and shoots, which was further validated through analysis of the activity of the NtPIN4 promoter. We used CRISPR‐Cas9 technology to generate mutants for NtPIN4 and observed that both T₀ and T₁ plants had a significantly increased axillary bud growth phenotype, as compared with the wild‐type plants. Therefore, NtPIN4 offers an opportunity for studying auxin‐dependent branching processes.     

Determination of intrinsic monod kinetic parameters for two heterotrophic tetrachloroethene (PCE)‐respiring strains and insight into their application

A complete set of mathematically identifiable and meaningful kinetic parameters estimates is needed to accurately describe the activity of individual populations that dehalorespire tetrachloroethene (PCE) and other chlorinated ethenes. These data may be difficult to extract from the literature because kinetic parameter estimates obtained using mixed cultures may reflect the activity of multiple dehalorespiring populations, while those obtained at low initial substrate‐to‐biomass ratios (S₀/X₀) are influenced by culture history and are generally not relevant to other systems. This study focused on estimation of electron donor and acceptor utilization kinetic parameters for the heterotrophic dehalorespirers Desulfuromonas michiganensis strain BB1 and Desulfitobacterium sp. strain PCE1. Electron acceptor utilization kinetic parameters that are identifiable and independent of culture history, i.e., intrinsic, could be estimated at S₀/X₀ ≥ 10, with both concentrations expressed as chemical oxygen demand (COD). However, the parameter estimates did not accurately describe dechlorination kinetics at lower S₀/X₀ ratios. The maximum specific substrate utilization rates (q_max) and half‐saturation constants (K_S) for PCE and trichloroethene (TCE) estimated for the two heterotrophic strains are higher than the values reported for Dehalococcoides cultures. These results suggest that the natural niche of Dehalococcoides strains that can metabolize a range of chlorinated ethenes may be to respire dichloroethene and vinyl chloride produced by Desulfuromonas and Desulfitobacterium strains or other populations that dechlorinate PCE and TCE at faster rates. Few data exist on the electron donor utilization kinetics of heterotrophic dehalorespirers. The results of this study suggest that Desulfuromonas and Desulfitobacterium strains should be able to compete for organic electron donors with other heterotrophic populations in the subsurface. Biotechnol. Bioeng. 2009; 104: 301–311 © 2009 Wiley Periodicals, Inc.