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1.
Front Microbiol ; 10: 1433, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31316484

RESUMO

RNA-based biomarkers have been successfully detected at field sites undergoing in situ bioremediation, but the detection of expressed enzymes is a more direct way to prove activity for a particular biocatalytic process of interest since they provide evidence of potential in situ activity rather than simply confirming presence and abundance of genes in a given population by measurement of DNA copies using qPCR. Here we successfully applied shotgun proteomics to field samples from a trichloroethene (TCE)-contaminated industrial site in southern Ontario, Canada that had been bio-augmented with the commercially available KB-1TM microbial culture. The KB-1TM culture contains multiple strains of Dehalococcoides mccartyi (D. mccartyi) as well as an organohalide respiring Geobacter species. The relative abundances of specific enzymatic proteins were subsequently compared to corresponding qPCR-derived levels of DNA and RNA biomarkers in the same samples. Samples were obtained from two wells with high hydraulic connectivity to the KB-1TM-bioaugemented enhanced in situ bioremediation system, and two control wells that showed evidence of low levels of native organohalide respiring bacteria (OHRB), Dehalococcoides and Geobacter. Enzymes involved in organohalide respiration were detected in the metaproteomes of all four field samples, as were chaperonins of D. mccartyi, chemotaxis proteins, and ATPases. The most highly expressed RDase in the bioaugmentation culture (VcrA) was the most highly detected enzyme overall in the bioaugmented groundwater samples. In one background groundwater well, we found high expression of the Geobacter pceA RDase. The DNA and RNA biomarkers detected using qPCR-based assays were a set of orthologs of Dehalococcoides reductive dehalogenases (VcrA, TceA, BvcA, dehalogenase "DET1545"), and the Ni-Fe uptake hydrogenase, HupL. Within a sample, RNA levels for key enzymes correlated with relative protein abundance. These results indicate that laboratory observations of TCE-bioremediation biomarker protein expression are recapitulated in field environmental systems and that both RNA and protein biomarker monitoring hold promise for activity monitoring of in situ populations of OHRB.

2.
Microorganisms ; 6(1)2018 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-29419787

RESUMO

Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10-12 to 5.9 × 10-10 microelectron equivalents per cell per hour (µeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R² = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration rates to decline immediately (>95% decline within one hour). Although transcript levels did decline, they did so more slowly than the respiration rate observed (transcript decay rates between 0.02 and 0.03 per hour). Data from strain-specific probes on the pangenome array strains suggest that a minor DMC strain in KB-1™ that harbors a bvcA homolog preferentially recovered following oxygen stress relative to the dominant, vcrA-containing strain.

3.
Appl Microbiol Biotechnol ; 99(5): 2313-27, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25467924

RESUMO

To better understand the quantitative relationships between messenger RNA (mRNA) and protein biomarkers relevant to bioremediation, we quantified and compared respiration-associated gene products in an anaerobic syntrophic community. Respiration biomarkers for Dehalococcoides, an organohalide reducer, and Methanospirillum, a hydrogenotrophic methanogen, were quantified via qRT-PCR for mRNA and multiple reaction monitoring (MRM) of proteotypic peptides for protein. mRNA transcripts of the Dehalococcoides reductive dehalogenases PceA, TceA, and DMC1545, and hydrogenase HupL, as well as the Methanospirillum oxidoreductases MvrD and FrcA were shown to be similarly regulated with respect to their temporal responses to substrate addition. However, MvrD was two orders of magnitude lower in mRNA abundance. Per cell, Dehalococcoides protein biomarkers quantified were more abundant than Methanospirillum proteins. Comparing mRNA with protein abundance, poor correlations were observed between mRNA transcript levels and the net protein produced. For example, Dehalococcoides HupL and TceA transcripts were similarly abundant though TceA was far more abundant at the protein level (167 ± 121 vs. 1095 ± 337 proteins per cell, respectively). In Methanospirillum, MvrD maintained comparable per-cell protein abundance to FrcA (42 ± 14 vs. 60 ± 1 proteins per cell, respectively) despite the significantly lower transcript levels. Though no variability in protein decay rates was observed, the mRNA translation rate quantified for TceA was greater than the other Dehalococcoides targets monitored. These data suggest that there is considerable variation in the relationship between mRNA abundance and protein production both across transcripts within an organism and across organisms. This highlights the importance of empirically based studies for interpreting biomarker levels in environmentally relevant organisms.


Assuntos
Proteínas de Bactérias/análise , Biomarcadores/análise , Biotransformação , Chloroflexi/metabolismo , Perfilação da Expressão Gênica , Methanospirillum/metabolismo , RNA Mensageiro/análise , Anaerobiose , Proteínas de Bactérias/genética , Chloroflexi/genética , Methanospirillum/genética , Consórcios Microbianos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
4.
Appl Environ Microbiol ; 80(19): 6062-72, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25063656

RESUMO

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).


Assuntos
Chloroflexi/genética , Regulação Bacteriana da Expressão Gênica , Hidrocarbonetos Clorados/metabolismo , Oxirredutases/genética , Transcriptoma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chloroflexi/enzimologia , Chloroflexi/fisiologia , Clorofenóis/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Hidrogenase/genética , Hidrogenase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Óperon/genética , Oxirredutases/metabolismo , Subunidades Proteicas
5.
Environ Sci Technol ; 47(8): 3724-33, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23363057

RESUMO

Bioremediation of chlorinated ethenes via anaerobic reductive dechlorination relies upon the activity of specific microbial populations--most notably Dehalococcoides (DHC) strains. In the lab and field Dehalococcoides grow most robustly in mixed communities which usually contain both fermenters and methanogens. Recently, researchers have been developing quantitative molecular biomarkers to aid in field site diagnostics and it is hoped that these biomarkers could aid in the modeling of anaerobic reductive dechlorination. A comprehensive biokinetic model of a community containing Dehalococcoides mccartyi (formerly D. ethenogenes) was updated to describe continuously fed reactors with specific biomass levels based on quantitative PCR (qPCR)-based population data (DNA and RNA). The model was calibrated and validated with subsets of chemical and molecular biological data from various continuous feed experiments (n = 24) with different loading rates of the electron acceptor (1.5 to 482 µeeq/L-h), types of electron acceptor (PCE, TCE, cis-DCE) and electron donor to electron acceptor ratios. The resulting model predicted the sum of dechlorination products vinyl chloride (VC) and ethene (ETH) well. However, VC alone was under-predicted and ETH was over predicted. Consequently, competitive inhibition among chlorinated ethenes was examined and then added to the model. Additionally, as 16S rRNA gene copy numbers did not provide accurate model fits in all cases, we examined whether an improved fit could be obtained if mRNA levels for key functional enzymes could be used to infer respiration rates. The resulting empirically derived mRNA "adjustment factors" were added to the model for both DHC and the main methanogen in the culture (a Methanosaeta species) to provide a more nuanced prediction of activity. Results of this study suggest that at higher feeding rates competitive inhibition is important and mRNA provides a more accurate indicator of a population's instantaneous activity than 16S rRNA gene copies alone as biomass estimates.


Assuntos
Chloroflexi/metabolismo , Halogenação , Hidrocarbonetos Halogenados/metabolismo , Hidrocarbonetos Halogenados/farmacocinética , Metano/metabolismo , Modelos Biológicos , Aerobiose , Biodegradação Ambiental , Biomarcadores/metabolismo , Biomassa , Chloroflexi/genética , Elétrons , Etilenos/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Tricloroetileno/metabolismo , Tricloroetileno/farmacocinética , Cloreto de Vinil/metabolismo
6.
Environ Sci Technol ; 47(1): 372-81, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23153046

RESUMO

Molecular biomarkers hold promise for inferring rates of key metabolic activities in complex microbial systems. However, few studies have assessed biomarker levels for simultaneously occurring (and potentially competing) respirations. In this study, methanogenesis biomarkers for Methanospirillum hungatei were developed, tested, and compared to Dehalococcoides mccartyi biomarkers in a well-characterized mixed culture. Proteomic analyses of mixed culture samples (n = 4) confirmed expression of many M. hungatei methanogenesis enzymes. The mRNAs for two oxidoreductases detected were explored as quantitative biomarkers of hydrogenotrophic methanogenesis: a coenzyme F(420)-reducing hydrogenase (FrcA) and an iron sulfur protein (MvrD). As shown previously in D. mccartyi, M. hungatei transcript levels correlated linearly with measured (R = 0.97 for FrcA, R = 0.91 for MvrD; n = 7) or calculated respiration rate (R = 0.81 for FrcA, R = 0.62 for MvrD; n = 35) across two orders of magnitude on a log-log scale. The average abundance of MvrD transcripts was consistently two orders of magnitude lower than FrcA, regardless of experimental condition. In experiments where M. hungatei was competing for hydrogen with D. mccartyi, transcripts for the key respiratory hydrogenase HupL were generally less abundant per mL than FrcA and more abundant than MvrD. With no chlorinated electron acceptor added, HupL transcripts fell below both targets. These biomarkers hold promise for the prediction of in situ rates of respiration for these microbes, even when growing in mixed culture and utilizing a shared substrate which has important implications for both engineered and environmental systems. However, the differences in overall biomarker abundances suggest that the strength of any particular mRNA biomarker relies upon empirically established quantitative trends under a range of pertinent conditions.


Assuntos
Metano/metabolismo , Methanospirillum/fisiologia , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Bactérias/metabolismo , Biomarcadores/metabolismo , Chloroflexi/fisiologia , Expressão Gênica , Hidrocarbonetos Clorados/metabolismo , Hidrogênio/metabolismo , Oxirredutases/metabolismo , Proteômica
7.
Environ Sci Technol ; 46(17): 9388-97, 2012 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-22812668

RESUMO

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 culture's maximum respiration rate. The dehalogenase TceA and the Ni-Fe hydrogenase HupL transcripts were positively correlated (Pearson's 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.


Assuntos
Proteínas de Bactérias/metabolismo , Chloroflexi/metabolismo , Etilenos/metabolismo , Hidrocarbonetos Clorados/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Bactérias/genética , Chloroflexi/enzimologia , Chloroflexi/genética , Poluentes Ambientais/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Mensageiro/genética
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