Ruth E. Richardson

Comparative Proteomics of Dehalococcoides spp. Reveals Strain-Specific Peptides Associated with Activity

ABSTRACT Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.

Temporal Expression of Respiratory Genes in an Enrichment Culture Containing Dehalococcoides ethenogenes

ABSTRACT Multiple reductive dehalogenase (RDase), hydrogenase (H2ase), and other respiration-associated (RA) oxidoreductase genes have been identified in cultured representatives of Dehalococcoides. Although their products are likely to play key roles in the environmentally important process of reductive dechlorination, very little information is available about their regulation and specific functions. Here we show increased expression and temporal variability in the expression of five RDase genes and in the expression of genes for a putative formate dehydrogenase (Fdh) and two H2ases, including a periplasmic [Ni/Fe] H2ase (Hup) and a cytoplasmic [Fe] H2ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H2ase Hup, and the RDase targets TceA and DET0162 were expressed most highly, with average levels 34 (± 7.5)-, 23 (± 6.7)-, 16 (± 3.3)-, and 13 (± 3.3)-fold higher, respectively, than that for RNA polymerase (RpoB). H2ase and RA transcripts reached their respective expression maxima within the first 2 h after feeding. RDase transcripts, however, were most highly expressed after 3 h and exhibited greater temporal variability than other transcripts. Comparison with D. ethenogenes strain 195 pure culture expression levels indicated that RDase DET1545 was more highly expressed in mixed cultures, where, on average, its transcript level was sixfold higher than that of RpoB. While the specific functions of several of these gene products remain elusive, the high expression levels and temporal variability reported here suggest that these groups of enzymes are metabolically important for the respiration of chlorinated ethenes in mixed cultures containing Dehalococcoides.

Comparative metagenomics of three Dehalococcoides-containing enrichment cultures: the role of the non-dechlorinating community

BackgroundThe Dehalococcoides are strictly anaerobic bacteria that gain metabolic energy via the oxidation of H2 coupled to the reduction of halogenated organic compounds. Dehalococcoides spp. grow best in mixed microbial consortia, relying on non-dechlorinating members to provide essential nutrients and maintain anaerobic conditions.A metagenome sequence was generated for the dechlorinating mixed microbial consortium KB-1. A comparative metagenomic study utilizing two additional metagenome sequences for Dehalococcoides-containing dechlorinating microbial consortia was undertaken to identify common features that are provided by the non-dechlorinating community and are potentially essential to Dehalococcoides growth.ResultsThe KB-1 metagenome contained eighteen novel homologs to reductive dehalogenase genes. The metagenomes obtained from the three consortia were automatically annotated using the MG-RAST server, from which statistically significant differences in community composition and metabolic profiles were determined. Examination of specific metabolic pathways, including corrinoid synthesis, methionine synthesis, oxygen scavenging, and electron-donor metabolism identified the Firmicutes, methanogenic Archaea, and the ∂-Proteobacteria as key organisms encoding these pathways, and thus potentially producing metabolites required for Dehalococcoides growth.ConclusionsComparative metagenomics of the three Dehalococcoides-containing consortia identified that similarities across the three consortia are more apparent at the functional level than at the taxonomic level, indicating the non-dechlorinating organisms’ identities can vary provided they fill the same niche within a consortium. Functional redundancy was identified in each metabolic pathway of interest, with key processes encoded by multiple taxonomic groups. This redundancy likely contributes to the robust growth and dechlorination rates in dechlorinating enrichment cultures.

Genomic insights into organohalide respiration

In the last few years there has been a burst of genomes released for organohalide respiring bacteria (referred to as OHRB herein though the process is otherwise known as dehalorespiration, reductive dechlorination, or halorespiration). The microorganisms are employed in bioremediation of sites contaminated with chlorinated ethene, ethane, and methanes, as well as chlorinated aromatics. Of particular note are the releases of the first Dehalogenimonas genome (a Dehalococcoides-related Chloroflexi) and not one but seven Dehalobacter (meta)genomes. Collectively, genomes from these three genera (Dehalococcoides, Dehalogenimonas, and Dehalobacter) clearly support their niche as obligate OHRB, while other genera with sequenced genomes (Desulfitobacterium, Geobacter, and Anaeromyxobacter) maintain organohalide respiration (OHR) as one of many possible energy conserving respiration strategies. The obligate OHRB genomes consistently harbor 10-39 unique reductive dehalogenase (RDase) genes and they are flanked with not only transcriptional regulators but also transposition related genes. Active transposition likely plays a key role in the accumulation of such a broad and tightly regulated dehalogenase repertoire. Functional assays are now the bottleneck for genome-informed discovery of dehalogenase substrate ranges.

Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated "Dehalococcoides" and Methanospirillum Species†

ABSTRACT This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming “Dehalococcoides ethenogenes” strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of “Dehalococcoides” and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole-sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple-reaction monitoring (MRM) assays using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R(2) > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 +/- 1.7 x 10(3) proteins cell(-1) in the KB1 bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.

Microbial activity in soils following steam treatment

Steam enhanced extraction (SEE) is an aquifer remediation technique that can be effective at removing the bulk of non-aqueous phase liquid (NAPL) contamination from the subsurface, particularly highly volatile contaminants. However, low volatility compounds such as polynuclear aromatic hydrocarbons (PAHs) are less efficientlyremoved by this process. This research evaluated the effects of steam injection on soilmicrobial activity, community structure, and the potential for biodegradation of contaminants following steam treatment. Three different soils were evaluated: alaboratory-prepared microbially-enriched soil, soil from a creosote contaminated fieldsite, and soil from a chlorinated solvent and waste oil contaminated field site. Results from field-scale steaming are also presented. Microbial activity before and after steam treatment was evaluated using direct epifluorescent microscopy (DEM) using the respiratory activity dye 5-cyano-2,3, ditolyl tetrazolium chloride (CTC) in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) to yield a quantitative assessment of active and total microbial numbers. DEM results indicate that steamed soils that were analyzed while still hot exhibited microbial activity levels that were below detection. However, soil samples that were slowly cooled, more closely reflecting the conditions of applied SEE, exhibited microbial activity levels that were comparable to presteamed soils. Samples from a field-site where steam was applied continuously for 6 weeks also showed high levels of microbial activity following cooling. The metabolic capabilities of the steamed communities were investigated by measuring cell growth in enrichment cultures on various substrates. These studies provided evidence that organisms capable of biodegradation were among the mesophilic populations that survived steam treatment. Fluorescent in situ hybridization (FISH) analysis of the soils with domain-level rRNA probes suggest that both Archaea and Bacteria survived steam exposure.

Quantitative estimation of Dehalococcoides mccartyi at laboratory and field scale: Comparative study between CARD-FISH and Real Time PCR

Dehalococcoides mccartyi detectability in the field is a fundamental tool to assess the efficiency of natural attenuation or engineered bioremediation in chlorinated solvent-contaminated sites. This study reports on the direct comparison of quantitative data obtained by Real Time PCR (qPCR) and CAtalyzed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH) over a wide range of Dehalococcoides concentrations (10-10(8) cells mL(-1)) both in three independent 10-fold serial dilutions of a laboratory dechlorinating enrichment and in 49 groundwater samples from 6 different contaminated sites. Dehalococcoides enumeration by CARD-FISH yielded a linear curve in the analyzed concentration range which was consistent with the expected concentrations and showed good reproducibility in triplicate assays. Alternatively, qPCR did not allow for the discrimination of 16S rRNA gene concentrations lower than 10(3) gene copies mL(-1) either in the dechlorinating mixed culture or in field samples. Overall this study highlights the limits of qPCR quantification, especially in samples where low concentrations of this microorganism may be expected, and suggests the use of a confirmatory methodology under these particular conditions.

Relating chloroethene respiration rates in Dehalococcoides to protein and mRNA biomarkers.

Molecular biomarkers could provide critical insight into myriad in situ microbial activities. In this study we explore correlations of both mRNA and protein biomarkers with chloroethene respiration rate in Dehalococcoides. In a series of continuously fed dechlorinating mixed-culture microcosm experiments (n = 26), we varied respiratory substrates, substrate ratios and feeding rates. Transcript levels for most biomarkers were responsive down to 0.01× the cultures maximum respiration rate. The dehalogenase TceA and the Ni-Fe hydrogenase HupL transcripts were positively correlated (Pearsons r of 0.89 and 0.88, respectively) with respiration rates on log-log plots between 1.5 and 280 μeeq/L-hr for mRNA abundances of 10(7) to 10(10) transcripts/mL (0.07-230 transcripts/genome). These trends were independent of the types of chloroethene or electron donors fed. Other mRNA target levels plateaued or declined at respiration rates above 5 μeeq/L-hr. Using both relative and absolute protein quantification methods, we found that per-genome protein abundances of most targeted biomarkers did not statistically change over the experimental time frames. However, quantified enzyme levels allowed us to calculate in vivo enzyme-specific rate constants (k(cat)) for the dehalogenases PceA and TceA: 400 and 22 substrate molecules/enzyme-sec, respectively. Overall, these data support the promise of both mRNA and protein biomarkers for estimating process rates through either empirical (mRNA-based) or kinetic (protein-based) models, but they require follow-up studies in other cultures and at active remediation sites.

Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

ABSTRACT A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ± 0.6 μM Cl−/h)- and fast (22.9 ± 9.6 μM Cl−/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n = 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112).