Metagenome-Guided Proteomic Quantification of Reductive Dehalogenases in the Dehalococcoides mccartyi-Containing Consortium SDC-9

Abstract

At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). Metagenome sequencing of the commercial, Dhccontaining consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13 C-/ 15 N-labeled peptides determined 1.8 × 10 3 TceA and 1.2 × 10 2 VcrA RDase molecules per Dhc cell. The MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

Respiratory Vinyl Chloride Reductive Dechlorination to Ethene in TceA-Expressing Dehalococcoides mccartyi

“…have to scavenge adequate amounts of complete cobamides or precursor molecules to enable the maturation of RDases for energy conservation. For laboratory cultivation of Dehalococcoidia isolates, B 12 has been commonly supplied in the range of 25–50 μg L –1 . ,,− During growth with TCE, targeted proteomic analysis quantified averages of 7.6 × 10 3 and 1.8 × 10 3 TceA proteins per Dhc cell in consortium KB-1 and consortium SDC-9, respectively. , Assuming each TceA protein contains one cobamide molecule (i.e., a 1:1 ratio), , the theoretical B 12 demand to reach the observed cell densities (Table ) would be 0.5–2.4 and 0.3–2.7 μg L –1 for Dhc strain FL2 and strain 195, respectively (eq ). …”

“…Although data for corrinoid types and concentrations in groundwater aquifers are not available, the indigenous corrinoid pool is unlikely to meet Dhc strains’ μg L –1 level B 12 demand to achieve maximum growth yields and dechlorination activity. Groundwater monitoring data indicated that meaningful in situ dechlorination activity and ethene formation can be achieved when Dhc cell titers exceed 10 6 L –1 . , Assuming 1.8 × 10 3 to 7.6 × 10 3 RDase (e.g., TceA) proteins per Dhc cell , and a cobamide-to-RDase molar ratio of 1:1, 8.1 to 34 pg L –1 of B 12 should theoretically meet the corrinoid demand of 10 6 Dhc cells L –1 and allow in situ bioremediation to take place. Therefore, it is conceivable that in situ aquifer conditions exist that sustain Dhc reductive dechlorination activity even at meager (i.e., ng L –1 ) corrinoid pool sizes.…”

“…32,46,50−53 During growth with TCE, targeted proteomic analysis quantified averages of 7.6 × 10 3 and 1.8 × 10 3 TceA proteins per Dhc cell in consortium KB-1 and consortium SDC-9, respectively. 54,55 Assuming each TceA protein contains one cobamide molecule (i.e., a 1:1 ratio), 56,57 the theoretical B 12 demand to reach the observed cell densities (Table 1) would be 0.5−2.4 and 0.3− 2.7 μg L −1 for Dhc strain FL2 and strain 195, respectively (eq 1).…”

Metagenomic Analysis Reveals Microbial Interactions at the Biocathode of a Bioelectrochemical System Capable of Simultaneous Trichloroethylene and Cr(VI) Reduction

“…To this aim, metagenomics approaches provide reliable information about the key enzymes/genes involved in the degradation and detoxification of environmental pollutants to understand better the microbial community structure, functions, and interactions in contaminated matrices or engineered systems for the biological removal of pollutants, with implications for process optimization and bioremediation application at field scale ( Bharagava et al, 2019 ; Lapidus and Korobeynikov, 2021 ). Metagenomic studies have been previously conducted on stable dechlorinating consortia to shed light on the metabolic features of the microbial components and their role in the RD processes under different conditions ( Brisson et al, 2012 ; Maphosa et al, 2012 ; Dam et al, 2017 ; Wang et al, 2019 ; Kucharzyk et al, 2020 ). Similarly, some metagenomic studies have also been conducted on biological systems for Cr(VI) removal to explore the metabolic potential, including Cr(VI) remediation genes, primarily in wastewater treatment systems or microbial consortia ( Miao et al, 2015 ; Sun et al, 2019 ; Pei et al, 2020 ; Rahman and Thomas, 2021 ).…”

Synergistic Zerovalent Iron (Fe0) and Microbiological Trichloroethene and Perchlorate Reductions Are Determined by the Concentration and Speciation of Fe

“…SDC-9 culture contains strains of D. mccartyi with pceA , tceA , and vcrA reductive dehalogenase genes. , …”