MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation.

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM.IMPORTANCEDichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

Unveiling microbial community structure and metabolic pathway over carbon cloth-titanium nitride-polyaniline biocathode for effective dichloromethane transformation.

Chlorinated volatile organic compounds (Cl-VOCs) have dramatically biotoxicity and environmental persistence due to the presence of chlorine atoms, seriously jeopardizing ecological security and human health. Dichloromethane (DCM) as a model pollutant, is widely applied in solvents, extractants and cleaning agents in the pharmaceutical, chemical and food industries. In this study, highly biocompatible and conductive carbon cloth-titanium nitride-polyaniline (CC-TiN-PANI) bioelectrodes were obtained for DCM degradation in microbial electrolysis cell (MEC). The good adhesion of TiN and PANI on the electrode surface was demonstrated. The degradation kinetics were fitted by the Haldane model, compared to the CC bioelectrode (0.8 h-1), the proportion of maximum degradation rates to half-saturation concentration (Vmax/Km) of CC-TiN (1.4 h-1) and CC-TiN-PANI (2.2 h-1) bioelectrodes were enhanced by 1.8 and 2.8 times, respectively. Microbial community structure analysis illuminated that the dominant genera on the biofilm were Alicycliphilus and Hyphomicrobium, and the abundance was enhanced significantly with the modification of TiN and PANI. The dechlorination of DCM to formaldehyde could be catalyzed by DCM dehalogenase (DcmA) or by haloalkane dehalogenase (DhlA). And further oxidized to formate: 1) direct catalyzed by formaldehyde dehydrogenase (FdhA); 2) conjugated with glutathione by S-(hydroxymethyl)-glutathione synthase (Gfa), S-(hydroxymethyl)-glutathione dehydrogenase (FrmA) and S-formyl-glutathione hydrolase (FrmB); 3) conjugation with tetrahydrofolate (H4F) and/or tetrahydromethanopterin.

Mobile genetic elements associated with utilization of dichloromethane and methanol as energy sources in Cupriavidus metallidurans.

Cupriavidus metallidurans strain PD11 isolated from laboratory waste drainage can use C1 compounds, such as dichloromethane (DCM) and methanol, as a sole carbon and energy source. However, strain CH34 (a type-strain) cannot grow in the medium supplemented with DCM. In the present study, we aimed to unravel the genetic elements underlying the utilization of C1 compounds by strain PD11. The genome subtraction approach indicated that only strain PD11 had several genes highly homologous to those of Herminiimonas arsenicoxydans strain ULPAs1. Moreover, a series of polymerase chain reaction (PCR) to detect the orthologs of H. arsenicoxydans genes and the comparative study of the genomes of three strains revealed that the 87.9 kb DNA fragment corresponding to HEAR1959 to HEAR2054 might be horizontally transferred to strain PD11. The 87.9 kb DNA fragment identified was found to contain three genes whose products were putatively involved in the metabolism of formaldehyde, a common intermediate of DCM and methanol. In addition, reverse transcription PCR analysis showed that all three genes were significantly expressed when strain PD11 was cultivated in the presence of DCM or methanol. These findings suggest that strain PD11 can effectively utilize the C1 compounds because of transfer of the mobile genetic elements from other bacterial species, for instance, from H. arsenicoxydans.

From mec cassette to rdhA: a key Dehalobacter genomic neighborhood in a chloroform and dichloromethane-transforming microbial consortium.

Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes such as methanogenesis. Anaerobic biotransformation of CF and DCM has been well documented but typically independently of one another. CF is the electron acceptor for certain organohalide-respiring bacteria that use reductive dehalogenases (RDases) to dechlorinate CF to DCM. In contrast, known DCM degraders use DCM as their electron donor, which is oxidized using a series of methyltransferases and associated proteins encoded by the mec cassette to facilitate the entry of DCM to the Wood-Ljungdahl pathway. The SC05 culture is an enrichment culture sold commercially for bioaugmentation, which transforms CF via DCM to CO2. This culture has the unique ability to dechlorinate CF to DCM using electron equivalents provided by the oxidation of DCM to CO2. Here, we use metagenomic and metaproteomic analyses to identify the functional genes involved in each of these transformations. Though 91 metagenome-assembled genomes were assembled, the genes for an RDase-named acdA-and a complete mec cassette were found to be encoded on a single contig belonging to Dehalobacter. AcdA and critical Mec proteins were also highly expressed by the culture. Heterologously expressed AcdA dechlorinated CF and other chloroalkanes but had 100-fold lower activity on DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM and subsequent mineralization of DCM using the mec cassette. IMPORTANCE: Chloroform (CF) and dichloromethane (DCM) are regulated groundwater contaminants. A cost-effective approach to remove these pollutants from contaminated groundwater is to employ microbes that transform CF and DCM as part of their metabolism, thus depleting the contamination as the microbes continue to grow. In this work, we investigate bioaugmentation culture SC05, a mixed microbial consortium that effectively and simultaneously degrades both CF and DCM coupled to the growth of Dehalobacter. We identified the functional genes responsible for the transformation of CF and DCM in SC05. These genetic biomarkers provide a means to monitor the remediation process in the field.

Efficient degradation of aqueous dichloromethane by an enhanced microbial electrolysis cell: Degradation kinetics, microbial community and metabolic mechanisms.

Dichloromethane (DCM) has been listed as a toxic and harmful water pollutant, and its removal needs attention. Microbial electrolysis cells (MECs) are viewed as a promising alternative for pollutant removal, which can be strengthened from two aspects: microbial inoculation and acclimation. In this study, the MEC for DCM degradation was inoculated with the active sludge enhanced by Methylobacterium rhodesianum H13 (strain H13) and then acclimated in the form of a microbial fuel cell (MFC). Both the introduction of strain H13 and the initiation in MFC form significantly promoted DCM degradation. The degradation kinetics were fitted by the Haldane model, with Vmax, Kh, Ki and vmax values of 103.2 mg/L/hr, 97.8 mg/L, 268.3 mg/L and 44.7 mg/L/hr/cm2, respectively. The cyclic voltammogram implies that DCM redox reactions became easier with the setup of MEC, and the electrochemical impedance spectrogram shows that the acclimated and enriched microbes reduced the charge transfer resistance from the electrode to the electrolyte. In the biofilm, the dominant genera shifted from Geobacter to Hyphomicrobium in acclimation stages. Moreover, Methylobacterium played an increasingly important role. DCM metabolism mainly occurred through the hydrolytic glutathione S-transferase pathway, given that the gene dcmA was identified rather than the dhlA and P450/MO. The exogenous electrons facilitated the reduction of GSSG, directly or indirectly accelerating the GSH-catalyzed dehalogenation. This study provides support for the construction of an efficient and stable MEC for DCM removal in water environment.

Evaluation of alternate hosts for recombinant expression of a reductive dehalogenase.

Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl3, Na2S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in S. blattae, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H2-oxidizing C. necator H16, a novel host for the expression of RDases.

A Multifunctional Dehalobacter? Tandem Chloroform and Dichloromethane Degradation in a Mixed Microbial Culture.

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world but can be cleaned up through bioremediation. Although several strains of Dehalobacter restrictus can reduce CF to DCM and multiple Peptococcaceae can ferment DCM, these processes cannot typically happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here, we present a mixed microbial culture that can simultaneously metabolize CF and DCM and create an additional enrichment culture fed only DCM. Through genus-specific quantitative polymerase chain reaction, we find that Dehalobacter grows while either CF alone or DCM alone is converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without the addition of an exogenous electron donor, and through electron balance calculations, we show that DCM metabolism would produce sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and "self-feeding" could prolong bioremediation activity long after donor addition ends. Overall, understanding this mechanism informs strategies for culture maintenance and scale-up and benefits contaminated sites where the culture is employed for remediation worldwide.

Psidium guajava L.- dichloromethane and ethyl acetate fractions ameliorate striped catfish (Pangasianodon hypophthalmus) status via immune response, inflammatory, and apoptosis pathways.

Psidium guajava L. is known to possess immune-modulatory properties in humans and other mammals. Although the positive effects of P. guajava-based diets on the immunological status have been shown for some fish species, the underlying molecular mechanisms of its protective effects remain to be investigated. The aims of this study were to evaluate the immune-modulatory effects of two guava fractions from dichloromethane (CC) and ethyl acetate (EA) on striped catfish with in vitro and in vivo experiments. Striped catfish head kidney leukocytes were stimulated with 40, 20, 10 and 0 µg/ml of each extract fraction, and the immune parameters (ROS, NOS, and lysozyme) were examined at 6 and 24 h post stimulation. A final concentration of each fraction at 40, 10 and 0 µg/fish was then intraperitoneally injected into the fish. After 6, 24, and 72 h of administration, immune parameters as well as the expression of some cytokines related to innate and adaptive immune responses, inflammation, and apoptosis were measured in the head kidney. Results indicated that the humoral (lysozyme) and cellular (ROS and NOS) immune endpoints were regulated differently by CC and EA fractions depending on dose and time in both, in vitro and in vivo experiments. With regards to the in vivo experiment, the CC fraction of the guava extract could significantly enhance the TLRs-MyD88-NF-κB signaling pathway by upregulating its cytokine genes (tlr1, tlr4, myd88, and traf6), following the upregulation of inflammatory (nfκb, tnf, il1ß, and il6) and apoptosis (tp53 and casp8) genes 6 h after injection. Moreover, fish treated with both CC and EA fractions significantly enhanced cytokine gene expression including lys and inos at the later time points - 24 h or 72 h. Our observations suggest that P. guajava fractions modulate the immune, inflammatory, and apoptotic pathways.

Amburana cearensis seed extract stimulates astrocyte glutamate homeostatic mechanisms in hippocampal brain slices and protects oligodendrocytes against ischemia.

BACKGROUND: Stroke is a leading cause of death and disability worldwide. A major factor in brain damage following ischemia is excitotoxicity caused by elevated levels of the neurotransmitter glutamate. In the brain, glutamate homeostasis is a primary function of astrocytes. Amburana cearensis has long been used in folk medicine and seed extract obtained with dichloromethane (EDAC) have previously been shown to exhibit cytoprotective activity in vitro. The aim of the present study was to analyse the activity of EDAC in hippocampal brain slices. METHODS: We prepared a dichloromethane extract (EDAC) from A. cearensis seeds and characterized the chemical constituents by 1H and 13C-NMR. Hippocampal slices from P6-8 or P90 Wistar rats were used for cell viability assay or glutamate uptake test. Hippocampal slices from P10-12 transgenic mice SOX10-EGFP and GFAP-EGFP and immunofluorescence for GS, GLAST and GLT1 were used to study oligodendrocytes and astrocytes. RESULTS: Astrocytes play a critical role in glutamate homeostasis and we provide immunohistochemical evidence that in excitotoxicity EDAC increased expression of glutamate transporters and glutamine synthetase, which is essential for detoxifying glutamate. Next, we directly examined astrocytes using transgenic mice in which glial fibrillary acidic protein (GFAP) drives expression of enhanced green fluorescence protein (EGFP) and show that glutamate excitotoxicity caused a decrease in GFAP-EGFP and that EDAC protected against this loss. This was examined further in the oxygen-glucose deprivation (OGD) model of ischemia, where EDAC caused an increase in astrocytic process branching, resulting in an increase in GFAP-EGFP. Using SOX10-EGFP reporter mice, we show that the acute response of oligodendrocytes to OGD in hippocampal slices is a marked loss of their processes and EDAC protected oligodendrocytes against this damage. CONCLUSION: This study provides evidence that EDAC is cytoprotective against ischemia and glutamate excitotoxicity by modulating astrocyte responses and stimulating their glutamate homeostatic mechanisms.

Compartment-related aspects of XoxF protein functionality in Methylorubrum extorquens DM4 analysed using its cytoplasmic targeting.

The impact of periplasmic localisation on the functioning of the XoxF protein was evaluated in the well-studied dichloromethane-utilising methylotroph Methylorubrum extorquens DM4, which harbors only one paralogue of the xoxF gene. It was found that the cytoplasmic targeting of XoxF by expression of the corresponding gene without the sequence encoding the N-terminal signal peptide does not impair the activation and lanthanide-dependent regulation of the MxaFI-methanol dehydrogenase genes. Analysis of the viability of ΔxoxF cells complemented with the full-length and truncated xoxF gene also showed that the expression of cytoplasmically targeted XoxF even increases the resistance to acids. These results contradict the proposed function of the XoxF protein as an extracytoplasmic signal sensor. At the same time, the observed dynamics of growth with methanol, as well as with dichloromethane of strains expressing cytoplasmic-targeted XoxF, indicate the probable enzymatic activity of lanthanide-dependent methanol dehydrogenase in this compartment. Herewith, the only available substrate for this enzyme in cells growing with dichloromethane was formaldehyde, which is produced during the primary metabolism of the mentioned halogenated toxicant directly in the cytosol. These findings suggest that the maturation of XoxF-methanol dehydrogenase may occur already in the cytoplasm, while the factors changing affinity of this enzyme for formaldehyde are apparently absent there. Together with the demonstrated functioning of an enhancer-like upstream activating sequence in the promoter region of the xoxF gene in M. extorquens DM4, the obtained information enriches our understanding of the regulation, synthesis and role of the XoxF protein.

Identification and widespread environmental distribution of a gene cassette implicated in anaerobic dichloromethane degradation.

Anthropogenic activities and natural processes release dichloromethane (DCM, methylene chloride), a toxic chemical with substantial ozone-depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial species revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising 8-10 genes encoding proteins with 79.6%-99.7% amino acid identities. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system, and shotgun proteomics applied to two DCM-catabolizing cultures revealed high expression of proteins encoded on the mec gene cluster during anaerobic growth with DCM. In a DCM-contaminated groundwater plume, the abundance of mec genes strongly correlated with DCM concentrations (R2  = 0.71-0.85) indicating their potential value as process-specific bioremediation biomarkers. mec gene clusters were identified in metagenomes representing peat bogs, the deep subsurface, and marine ecosystems including oxygen minimum zones (OMZs), suggesting a capacity for DCM degradation in diverse habitats. The broad distribution of anaerobic DCM catabolic potential infers a role for DCM as an energy source in various environmental systems, and implies that the global DCM flux (i.e., the rate of formation minus the rate of consumption) might be greater than emission measurements suggest.

Anaerobic Biodegradation of Chloroform and Dichloromethane with a Dehalobacter Enrichment Culture.

Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. Complete dechlorination of CF has been reported under anaerobic conditions by microbes that respire CF to DCM and others that biodegrade DCM. The objectives of this study were to ascertain if a commercially available bioaugmentation enrichment culture (KB-1 Plus CF) uses an oxidative or fermentative pathway for biodegradation of DCM and to determine if the products from DCM biodegradation can support organohalide respiration of CF to DCM in the absence of an exogenous electron donor. In various treatments with the KB-1 Plus CF culture to which 14C-CF was added, the predominant product was 14CO2, indicating that oxidation is the predominant pathway for DCM. Recovery of 14C-DCM when biodegradation was still in progress confirmed that CF first undergoes reductive dechlorination to DCM. 14C-labeled organic acids, including acetate and propionate, were also recovered, suggesting that synthesis of organic acids provides a sink for the electron equivalents from oxidation of DCM. When the biomass was washed to remove organic acids from prior additions of exogenous electron donor and only CF and DCM were added, the culture completely dechlorinated CF. The total amount of DCM added was not sufficient to provide the electron equivalents needed to reduce CF to DCM. Thus, the additional reducing power came via the DCM generated from CF reduction. Nevertheless, the rate of CF consumption was considerably lower compared to that of treatments that received an exogenous electron donor. IMPORTANCE Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. One way to address this problem is to add microbes to the subsurface that can biodegrade these compounds. While microbes are known that can accomplish this task, less is known about the pathways used under anaerobic conditions. Some use an oxidative pathway, resulting mainly in carbon dioxide. Others use a fermentative pathway, resulting in formation of organic acids. In this study, a commercially available bioaugmentation enrichment culture (KB-1 Plus CF) was evaluated using carbon-14 labeled chloroform. The main product formed was carbon dioxide, indicating the use of an oxidative pathway. The reducing power gained from oxidation was shown to support reductive dechlorination of CF to DCM. The results demonstrate the potential to achieve full dechlorination of CF and DCM to nonhazardous products that are difficult to identify in the field.

Mineralization versus fermentation: evidence for two distinct anaerobic bacterial degradation pathways for dichloromethane.

Dichloromethane (DCM) is an anthropogenic pollutant with ozone destruction potential that is also formed naturally. Under anoxic conditions, fermentation of DCM to acetate and formate has been reported in axenic culture Dehalobacterium formicoaceticum, and to acetate, H2 and CO2 in mixed culture RM, which harbors the DCM degrader 'Candidatus Dichloromethanomonas elyunquensis'. RM cultures produced 28.1 ± 2.3 µmol of acetate from 155.6 ± 9.3 µmol DCM, far less than the one third (i.e., about 51.9 µmol) predicted based on the assumed fermentation model, and observed in cultures of Dehalobacterium formicoaceticum. Temporal metabolite analyses using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy revealed that no 13C-labeled acetate was formed in 13C-DCM-grown RM cultures, indicating acetate was not a direct product of DCM metabolism. The data were reconciled with DCM mineralization and H2 consumption via CO2 reduction to acetate and methane by homoacetogenic and methanogenic partner populations, respectively. In contrast, Dehalobacterium formicoaceticum produced 13C-labeled acetate and formate from 13C-DCM, consistent with a fermentation pathway. Free energy change calculations predicted that organisms with the mineralization pathway are the dominant DCM consumers in environments with H2 <100 ppmv. These findings have implications for carbon and electron flow in environments where DCM is introduced through natural production processes or anthropogenic activities.

Molecular Docking and Site-Directed Mutagenesis of Dichloromethane Dehalogenase to Improve Enzyme Activity for Dichloromethane Degradation.

Dichloromethane (DCM) dehalogenase in bacterial cells can catalyze the degradation of deleterious DCM in environments. However, the utility of naturally occurring DCM dehalogenase is often limited due to low enzyme activity and content in living cells. In this study, the gene encoding DCM dehalogenase was cloned from Methylobacterium rhodesianum and overexpressed in Escherichia coli. Based on molecular docking analysis of DCM dehalogenase using DCM as the ligand, all of the target amino acid residues within substrate binding pocket and 10 conservative amino acid residues were individually mutated to Ala. After determination of activity, R120, L121, W128, and T146 were chosen for further saturation mutation. Results showed that dcmT146A, dcmT146R, and dcmT146Q have higher activities, whereas dcmL121A, dcmT146L, dcmL121Q, and dcmL121F have retained activities. Next, these seven mutants with a single mutation on amino acid residue were chosen for double mutation. It was found that the mutant of dcmL121A/T146R exhibits the highest activity increasing by 52.8% relative to wild type. Bioinformatic and experimental analyses revealed that the mutant variant dcmL121A/T146R bears the reduced steric hindrance in the active center with a decreased number of amino acid residues within binding pocket from 8 to 5 while overall hydrophilicity increased. In addition, the number of hydrophobic amino acid residues within substrate binding pocket increased while Km value decreased. It was speculated that all these changes in mutant variant dcmL121A/T146R may contribute to the increase in catalytic activity. It can be concluded that our goal-orientated manipulation through homology modeling, molecular docking, and site-directed mutagenesis is effective for improvement of DCM dehalogenase activity and investigation of correlation between structure and function.

Method Development for DNA and Proteome SIP Analysis of Activated Sludge for Anaerobic Dichloromethane Biodegradation.

Dichloromethane (DCM) is a toxic, dense non-aqueous phase liquid (DNAPL) that pollutes groundwater in all industrialized countries. Fortunately, DCM can be used as the sole source of energy and organic carbon by anaerobic microorganisms and be transformed to benign end products such as acetate, formate, and bicarbonate. However, knowledge around the phylogenetic diversity of anaerobic microorganisms capable of DCM metabolism is limited. The genes and enzymes involved and details of the reaction mechanism are not known. Stable isotope probing (SIP) is a technique used to identify microbes involved in assimilation of elements. The isotopically labeled substrate can be recovered in DNA and protein (i.e., DNA-SIP and protein-SIP) which enables identification of both the microbial taxa and their respective proteins involved in the substrate degradation. Therefore, by applying a combination of SIP techniques with molecular approaches (i.e., Illumina Miseq sequencing and metaproteomics), DCM degrading organisms can be identified and characterized in a manner independent of anaerobic enrichment cultures. In our research, activated sludge from wastewater treatment plant was fed with unlabeled and 13C-labeled DCM, respectively. Here, we provide protocols and technical notes for DNA and protein extraction from activated sludge and present analysis pipelines for downstream molecular techniques.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway.IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

Individual stages of bacterial dichloromethane degradation mapped by carbon and chlorine stable isotope analysis.

The fractionation of carbon and chlorine stable isotopes of dichloromethane (CH2Cl2, DCM) upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed. Isotope effects for individual steps of the multi-stage DCM degradation process, including transfer across the cell wall from the aqueous medium to the cell cytoplasm, dehalogenase binding, and catalytic reaction, were considered. The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of CCl bonds, and not by inflow of DCM into cells. Chlorine isotope effects of DCM dehalogenation were initially masked in high density cultures, presumably due to inverse isotope effects of non-specific DCM oxidation under conditions of oxygen excess. Glutathione cofactor supply remarkably affected the correlation of variations of DCM carbon and chlorine stable isotopes (Δδ13C/Δδ37Cl), increasing corresponding ratio from 7.2-8.6 to 9.6-10.5 under conditions of glutathione deficiency. This suggests that enzymatic reaction of DCM with glutathione thiolate may involve stepwise breaking and making of bonds with the carbon atom of DCM, unlike the uncatalyzed reaction, which is a one-stage process, as shown by quantum-chemical modeling.

Temporal relationship between emitted and endogenous floral scent volatiles in summer- and winter-blooming Jasminum species.

Jasminum spp. is cultivated for their fragrant flowers used in essential oil production and cosmetic uses. An attempt was made to study the temporal variations in floral scent volatiles composition including emitted, free endogenous and glycosyl-linked volatile compounds from two summer-blooming species namely, Jasminum auriculatum and Jasminum grandiflorum as well as from two winter-blooming species namely, Jasminum multiflorum and Jasminum malabaricum. The overall emitted volatile organic compounds (VOCs) were found to be highest when the matrix Porapak Q 80/100 was used with dichloromethane (DCM) as elution solvent. The floral volatile emission from bud to senescence exhibited nocturnal maxima pattern for both the summer-blooming species. Both the winter-blooming species emitted its highest concentration at noon. The free endogenous concentrations of all VOCs were low when corresponding emitted concentrations were high. Enzymatic treatment of petal extract revealed that several aromatic volatiles including aromatic alcohols and monoterpenols are synthesized and stored in the flowers as water-soluble glycosides; these compounds were shown to accumulate in higher amounts in flowers at late bud stage. These findings indicate the utilization of the precursors, i.e. the volatile-conjugates, through hydrolysis followed by their release as free-volatiles at flower opening stage. The outcome as a whole suggests a linkage among the temporal pattern of emitted volatiles, free-endogenous volatiles and glycoside-bound volatile compounds in all above studied Jasminum spp. and provided an overview of their floral volatilome.

Dichloromethane biodegradation in multi-contaminated groundwater: Insights from biomolecular and compound-specific isotope analyses.

Dichloromethane (DCM) is a widespread and toxic industrial solvent which often co-occurs with chlorinated ethenes at polluted sites. Biodegradation of DCM occurs under both oxic and anoxic conditions in soils and aquifers. Here we investigated in situ and ex situ biodegradation of DCM in groundwater sampled from the industrial site of Themeroil (France), where DCM occurs as a major co-contaminant of chloroethenes. Carbon isotopic fractionation (εC) for DCM ranging from -46 to -22‰ were obtained under oxic or denitrifying conditions, in mineral medium or contaminated groundwater, and for laboratory cultures of Hyphomicrobium sp. strain GJ21 and two new DCM-degrading strains isolated from the contaminated groundwater. The extent of DCM biodegradation (B%) in the aquifer, as evaluated by compound-specific isotope analysis (δ13C), ranged from 1% to 85% applying DCM-specific εC derived from reference strains and those determined in this study. Laboratory groundwater microcosms under oxic conditions showed DCM biodegradation rates of up to 0.1 mM·day-1, with concomitant chloride release. Dehalogenase genes dcmA and dhlA involved in DCM biodegradation ranged from below 4 × 102 (boundary) to 1 × 107 (source zone) copies L-1 across the contamination plume. High-throughput sequencing on the 16S rrnA gene in groundwater samples showed that both contaminant level and terminal electron acceptor processes (TEAPs) influenced the distribution of genus-level taxa associated with DCM biodegradation. Taken together, our results demonstrate the potential of DCM biodegradation in multi-contaminated groundwater. This integrative approach may be applied to contaminated aquifers in the future, in order to identify microbial taxa and pathways associated with DCM biodegradation in relation to redox conditions and co-contamination levels.

Synthesis and characterization of IUdR loaded PEG/PCL/PEG polymersome in mixed DCM/DMF solvent: Experimental and molecular dynamics insights into the role of solvent composition and star architecture in drug dispersion and diffusion.

Combined experimental and simulation investigations have provided molecular level insights into 5-iodo-2'-deoxyuridine (IUdR) loading behavior for the novel PEG/PCL/PEG polymersome-like carriers in mixed dichloromethane/N,N-dimethylformamide (DCM/DMF) solvent. As with the experiments, a novel approach was applied for layer by layer tailoring of polyethylene glycol (PEG) and polycaprolactone (PCL) as PEG/PCL/PEG copolymer on the surface of magnetite nanoparticles (MNPs) by click chemistry. Experimental results indicated that IUdR, as an anti-cancer drug, could be encapsulated up to 80% EE in this nanocarrier and could be in-vitro released up to 90% during 120h. Computational studies, on the other hand, provide molecular level insights into the optimal performance of the carrier in terms of drug "Dispersion" and "Diffusion" patterns in equimolar DCM/DMF solvent. Molecular dynamics simulations of the system in four distinct solvation scenarios including pure DCM, mixed DCM/DMF, pure DMF and water, have proven that while hydrophobic solvents give rise to better "dispersion" of drugs, hydrophilic solvents lead for drug molecules to penetrate into the carrier and improve "diffusion" properties. Optimal conditions for drug encapsulation, as also confirmed through experiments, was observed for mixed DCM/DMF solvent in terms of proper diffusion and well dispersion. While drug "aggregates" were observed in DCM, poorly stable drug molecules with lowered penetrations were observed in pure DMF. Proper release properties with IUdR molecules staying on the surface of the carrier was also observed in water. The interesting role of the star-linear architecture was further scrutinized through distinctions made through analysis of interactions between IUdR molecules with "inner" and "outer" PEG sections.