Isolation of two new Dehalococcoides mccartyi strains with dissimilar dechlorination functions and their characterization by comparative genomics via microarray analysis.

Microbial reductive dechlorination of trichloroethene (TCE) in groundwater often results in the accumulation of dichloroethenes (DCEs). Dehalococcoides mccartyi (Dhc) are the only known bacteria capable of dechlorination beyond DCE to non-toxic ethene. In this study, two newly isolated Dhc strains (11a and 11a5) with dissimilar functional abilities are described. Strain 11a reductively dechlorinates TCE, 1,1-DCE, cis-DCE, trans-DCE, and vinyl chloride (VC) to ethene, while strain 11a5 dechlorinates TCE and all three DCE isomers only to VC. Each of these dechlorination reactions are coupled to growth by these strains. The VC dechlorination rate of strain 11a occurs at a rate of 258 nmol per min per mg of protein, about two times faster than previously reported stains. Strain 11a possesses the vcrA gene while strain 11a5 contains the tceA gene. Strains 11a and 11a5 share 100% 16S rRNA gene sequence identity with previously sequenced Dhc strains BAV1 and CBDB1, placing it within the Pinellas subgroup, and 85.4% and 89.5% of all genes present in the CBDB1 and BAV1 genomes were detected in strains 11a and 11a5, respectively, using a custom-designed microarray targeting four sequenced Dhc strains. Genes that were not detected in strains 11a and 11a5 are mostly within the high plasticity regions or integrated elements of the sequenced strains. This study reports the functional description and comparative genomics of two additional Dhc isolates and provides evidence that the observed functional incongruence between the activity and core genome phylogenies of Dhc strains is likely driven by the horizontal transfer of key reductive dehalogenase-encoding genes.

Global gene expression of Dehalococcoides within a robust dynamic TCE-dechlorinating community under conditions of periodic substrate supply.

A microarray targeting four sequenced strains in the Dehalococcoides (Dhc) genus was used to analyze gene expression in a robust long-term trichloroethene (TCE)-degrading microbial community (designated ANAS) during feeding cycles that involve conditions of periodic substrate supply. The Dhc transcriptome was examined at three time-points throughout a batch feeding cycle: T1 (27 h) when TCE, dichloroethene (DCE), and vinyl chloride (VC) were present; T2 (54 h) when only VC remained; and T3 (13 days) when Dhc had been starved of substrate for 9 days. Ninety percent of the Dhc open reading frames (ORFs) that were detected in the ANAS DNA were found to be expressed as RNA sometime during the time course, demonstrating extraordinary utilization of the streamlined genome. Ninety-seven percent of these transcripts were differentially expressed during the time course indicating efficiency of transcription through regulation in Dhc. Most Dhc genes were significantly down-regulated at T3 , responding to a lack of substrate as would be expected. The tceA and vcrA genes, which code for proteins with known chlorinated ethene reduction functions, were highly expressed at both T1 and T2 , whereas two other putative reductive dehalogenase genes (DET0173 and DET1545) were most highly expressed at T2 , likely in response to the presence of VC. Hydrogenases were most highly expressed at T1 , reflecting their important role in accumulating electrons used to initiate reductive dechlorination and other biosynthesis pathways. Cobalamin transport genes were preferentially expressed at T2 , reflecting an increase in corrinoid transport as chloroethenes were degraded and a decrease in activity of the transport system after dehalogenation was complete. This is the first application of a microarray targeting a known genus, including both core genomes and identified strain-specific genes, to improve our understanding of transcriptional dynamics within an undefined microbial community.

Metagenomic analysis of a stable trichloroethene-degrading microbial community.

Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.

Sustainable syntrophic growth of Dehalococcoides ethenogenes strain 195 with Desulfovibrio vulgaris Hildenborough and Methanobacterium congolense: global transcriptomic and proteomic analyses.

Dehalococcoides ethenogenes strain 195 (DE195) was grown in a sustainable syntrophic association with Desulfovibrio vulgaris Hildenborough (DVH) as a co-culture, as well as with DVH and the hydrogenotrophic methanogen Methanobacterium congolense (MC) as a tri-culture using lactate as the sole energy and carbon source. In the co- and tri-cultures, maximum dechlorination rates of DE195 were enhanced by approximately three times (11.0±0.01 µmol per day for the co-culture and 10.1±0.3 µmol per day for the tri-culture) compared with DE195 grown alone (3.8±0.1 µmol per day). Cell yield of DE195 was enhanced in the co-culture (9.0±0.5 × 10(7) cells per µmol Cl(-) released, compared with 6.8±0.9 × 10(7) cells per µmol Cl(-) released for the pure culture), whereas no further enhancement was observed in the tri-culture (7.3±1.8 × 10(7) cells per µmol Cl(-) released). The transcriptome of DE195 grown in the co-culture was analyzed using a whole-genome microarray targeting DE195, which detected 102 significantly up- or down-regulated genes compared with DE195 grown in isolation, whereas no significant transcriptomic difference was observed between co- and tri-cultures. Proteomic analysis showed that 120 proteins were differentially expressed in the co-culture compared with DE195 grown in isolation. Physiological, transcriptomic and proteomic results indicate that the robust growth of DE195 in co- and tri-cultures is because of the advantages associated with the capabilities of DVH to ferment lactate to provide H(2) and acetate for growth, along with potential benefits from proton translocation, cobalamin-salvaging and amino acid biosynthesis, whereas MC in the tri-culture provided no significant additional benefits beyond those of DVH.

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.

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.

Phylogenetic analysis of TCE-dechlorinating consortia enriched on a variety of electron donors.

Two rapidly fermented electron donors, lactate and methanol, and two slowly fermented electron donors, propionate and butyrate, were selected for enrichment studies to evaluate the characteristics of anaerobic microbial consortia that reductively dechlorinate TCE to ethene. Each electron donor enrichment subculture demonstrated the ability to dechlorinate TCE to ethene through several serial transfers. Microbial community analyses based upon 16S rDNA, including terminal restriction fragment length polymorphism (T-RFLP) and clone library/sequencing, were performed to assess major changes in microbial community structure associated with electron donors capable of stimulating reductive dechlorination. Results demonstrated that five phylogenic subgroups or genera of bacteria were present in all consortia, including Dehalococcoides sp., low G+C Gram-positives (mostly Clostridium and Eubacterium sp.), Bacteroides sp., Citrobacter sp., and delta Proteobacteria (mostly Desulfovibrio sp.). Phylogenetic association indicates that only minor shifts in the microbial community structure occurred between the four alternate electron donor enrichments and the parent consortium. Inconsistent detection of Dehalococcoides spp. in clone libraries and T-RFLP of enrichment subcultures was resolved using quantitative polymerase chain reaction (Q-PCR). Q-PCR with primers specific to Dehalococcoides 16S rDNA resulted in positive detection of this species in all enrichments. Our results suggest that TCE-dechlorinating consortia can be stably maintained on a variety of electron donors and that quantities of Dehalococcoides cells detected with Dehalococcoides specific 16S rDNA primer/probe sets do not necessarily correlate well with solvent degradation rates.