Dehalococcoides spp. in river sediments: insights in functional diversity and dechlorination activity

In dit proefschrift staan Dehaloccoides spp. centraal vanwege hun vermogen één van deze gechloreerde verbindingen, hexachloorbenzeen (HCB), af te breken tot een verbinding met minder chlooratomen. HCB werd tot voor kort voornamelijk toegepast als fungicide en pesticide en kan bijvoorbeeld vrijkomen bij de productie van synthetisch rubber. Daarnaast wordt het gevormd als bijproduct tijdens de productie van oplosmiddelen en pesticiden. HCB is kankerverwekkend, giftig en hoopt zich op in ecosystemen. Tegenwoordig is het gebruik ervan binnen de E.U. dan ook verboden, maar omdat de stof erg moeilijk afbreekbaar is zal deze nog gedurende lange tijd worden teruggevonden in het milieu. Grootschalig onderzoek is gedaan naar de aanwezigheid, activiteit en het dechlorinerende vermogen van Dehalococcoides spp. in riviersedimenten en de bodems in uiterwaarden van verschillende Europese rivieren (Elbe, Donau, Maas, Ebro, Brevilles)

Physiological and enzymatic studies of respiration in Dehalococcoides species strain CBDB1

Die Dissertation beschreibt die physiologischen und enzymatischen Fähigkeiten von Dehalococcoides sp Stamm CBDB1. Wachstum des Stammes CBDB1 auf Grundlage von Dehalorespiration mit HCB und PeCB konnte gezeigt werden. Diese Ergebnisse stellen ein neues System für die Kultivierung von Stamm CBDB1 mit hochchlorierten Benzolen als Elektronenakzeptoren zur Verfügung. Hydrogenase und Dehalogenase sind die Schlüsselenzyme bei der Dehalorespiration. Beide Enzyme sind membrangebunden mit den katalytischen Zentren nach außen. Hydrogenaseaktivität konnte auch im Cytoplasma gemessen werden. Die Hydrogenase von Stamm CBDB1 ist sehr empfindlich gegenüber Sauerstoff, so verloren die Zellen sofort ihre Enzymaktivität, wenn sie der Luft ausgesetzt waren. Die Aktivität der reduktiven Dehalogenasen in Kulturen von Stamm CBDB1 angezüchet auf verschiedenen Chlorbenzolen weisen darauf hin, dass verschiedene Elektronenakzeptoren unter Umständen unterschiedliche reduktive Dehalogenasen induzieren. Die Dehalogenaseaktivität gemessen an ganzen Zellen mit artifiziellen Elektrondonoren lassen vermuten, dass ein Redoxpotential von ?-360 mV für die Reduktion von Chlorbenzolen benötigt wird. Auch sterische Effekte haben einem Einfluss auf die Dehalogenaseaktivität. So war die Dehalogenaseaktivität, die mit Methylviologen ( E0´=-450 mV) gemessen wurde, höher als die mit Ethylviologen ( E0´=-480 mV). Chinone scheinen als physiologische Elektronenmediatoren in der Transportkette von Stamm CBDB1 auszuscheiden, weil durch HONOQ, einen Inhibitor von Chinon abhängigen Redoxreaktionen, die Dechlorierung in intakten Zellen mit Wasserstoff als Elektronendonor nicht gehemmt werden konnte. Bei Dechlorierungsversuchen mit intakten Zellen und Wasserstoff als Elektronendonor in Gegenwart des Protonophors TCS stellte sich heraus, dass Stamm CBDB1 nicht auf einen reversen Elektronentransport angewiesen ist. 1,2,3,4-TeCB inhibierte die Dechlorierung in Zellkulturen mit Wasserstoff als Elektronendonor. Der genaue Mechanismus der Inhibition ist unbekannt. Es wird vermutet, dass 1,2,3,4-TeCB den Elektronentransport unterbricht, ohne mit der Hydrogenase oder Dehalogenase zu interagieren. Ein putatives Gencluster, bestehend aus den Strukturgenen hupS und hupL, die membrangebundene Gruppe 1 [Ni-Fe] Hydrogenasen codieren, wurde aus Stamm CBDB1 amplifiziert und sequenziert. Das Cluster enthielt außerdem hupD, ein Gen, das ein akzessorisches Protein für die Reifung der Hydrogenase codiert. Die Amplifizierung der drei Gene aus mRNA über RT-PCR bestätigte, dass das Gencluster als polycistronischer Messenger transkribiert wird. Dem amplifizierten Operon fehlte ein Gen, das für Cytochrom b codiert. Dieses Gen existiert in allen bisher bekannten membrangebundenen [Ni-Fe] Hydrogenasen, nicht aber in löslichen Hydrogenasen. Die Hydrogenaseaktivität wurde in der Membranfraktion gemessen. Ein einzigartiges hydrophobes Segment in der kleinen Untereinheit (HupS) der Hydrogenase könnte für das Anhaften an der Membran verantwortlich sein. An diesem Punkt unterscheidet sich das Hydrogenaseoperon von Stamm CBDB1 von den bisher in der Literatur beschriebenen.The thesis describes physiological properties of Dehalococcoides species strain DBDB1 and enzymes involved in dehalorespiration. Growth of strain CBDB1 with HCB and PeCB provided an efficient system for the cultivation with highly chlorinated benzenes as electron acceptors. The key enzymes, hydrogenase and dehalogenase activities, were membrane associated with catalytic sites oriented towards the outside. Hydrogenase activity was also detected in the cytoplasm. Hydrogenase of strain CBDB1 was found to be highly sensitive to oxygen. Reductive dehalogenase activity detected in cells of strain CBDB1 pregrown with different chlorobenzene congeners as electron acceptors indicated that the different electron acceptors might induce different reductive dehalogenases. Dehalogenase activity of whole cells detected with artificial electron donors indicated that a redox potential of £-360 mV is needed for the chlorobenzenes reduction. However, steric effects also influenced dehalogenase activity because a higher reaction rate of dehalogenase activity was measured with methyl viologen (Eo´= -450 mV) compared to ethyl viologen (Eo´= -480 mV). Quinones seem not to be physiological electron mediators in the electron transport of strain CBDB1, because HONOQ, an inhibitor of quinone dependent redox reactions, did not inhibit the reductive dechlorination reaction by intact cells with hydrogen as electron donor. Dechlorination by intact cells with hydrogen as electron donor in the presence of a protonophore, TCS, revealed that strain CBDB1 does not require reverse electron transport. 1,2,3,4-TeCB strongly inhibited the dechlorination by whole cells with hydrogen as electron donor. The precise mechanism of inhibition by 1,2,3,4-TeCB is unknown. However, 1,2,3,4-TeCB is believed to interfere somehow with a step in the electron transport of strain CBDB1 without inhibiting hydrogenase or dehalogenase activity. The putative gene cluster consisting of structural genes hupS and hupL coding for a membrane bound group-1 [Ni-Fe] hydrogenase was amplified and sequenced. The gene cluster also contained hupD, a gene encoding an accessory protein for hydrogenase maturation. Amplification of all three genes by RT-PCR from mRNA confirms that the investigated gene cluster is transcribed as a polycistronic messenger. The amplified operon lacks a gene coding for cytochrome b that is found in all other membrane bound [Ni-Fe] hydrogenases known so far, but is not present in soluble hydrogenases. However, based on the hydrogenase activity in the membrane fraction, a unique hydrophobic segment found in the small subunit (HupS) of the hydrogenase could be responsible for attaching the complex to the membrane. In this aspect, the hydrogenase operon of strain CBDB1 differs from all other membrane bound hydrogenase operons described so far

Localized Plasticity in the Streamlined Genomes of Vinyl Chloride Respiring Dehalococcoides

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism

Isolation and characterization of Dehalobacter sp. strain TeCB1 including identification of TcbA: A novel tetra- and trichlorobenzene reductive Dehalogenase

© 2017 Alfán-Guzmán, Ertan, Manefield and Lee. Dehalobacter sp. strain TeCB1 was isolated from groundwater near Sydney, Australia, that is polluted with a range of organochlorines. The isolated strain is able to grow by reductive dechlorination of 1,2,4,5-tetrachlorobenzene to 1,3- and 1,4-dichlorobenzene with 1,2,4-trichlorobenzene being the intermediate daughter product. Transient production of 1,2-dichlorobenzene was detected with subsequent conversion to monochlorobenzene. The dehalogenation capability of strain TeCB1 to respire 23 alternative organochlorines was examined and shown to be limited to the use of 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene. Growth on 1,2,4-trichlorobenzene resulted in the production of predominantly 1,3- and 1,4-dichlorobenzene. The inability of strain TeCB1 to grow on 1,2-dichlorobenzene indicated that the production of monochlorobenzene during growth on 1,2,4,5-tetarchlorobezene was cometabolic. The annotated genome of strain TeCB1 contained only one detectable 16S rRNA gene copy and genes for 23 full-length and one truncated Reductive Dehalogenase (RDase) homologs, five unique to strain TeCB1. Identification and functional characterization of the 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene RDase (TcbA) was achieved using native-PAGE coupled with liquid chromatography tandem mass spectrometry. Interestingly, TcbA showed higher amino acid identity with tetrachloroethene reductases PceA (95% identity) from Dehalobacter restrictus PER-K23 and Desulfitobacterium hafniense Y51 than with the only other chlorinated benzene reductase [i.e., CbrA (30% identity)] functionally characterized to date

New insights into Dehalococcoides mccartyi metabolism from a reconstructed metabolic network-based systems-level analysis of D. mccartyi transcriptomes

Organohalide respiration, mediated by Dehalococcoides mccartyi, is a useful bioremediation process that transforms ground water pollutants and known human carcinogens such as trichloroethene and vinyl chloride into benign ethenes. Successful application of this process depends on the fundamental understanding of the respiration and metabolism of D. mccartyi. Reductive dehalogenases, encoded by rdhA genes of these anaerobic bacteria, exclusively catalyze organohalide respiration and drive metabolism. To better elucidate D. mccartyi metabolism and physiology, we analyzed available transcriptomic data for a pure isolate (Dehalococcoides mccartyi strain 195) and a mixed microbial consortium (KB-1) using the previously developed pan-genome-scale reconstructed metabolic network of D. mccartyi. The transcriptomic data, together with available proteomic data helped confirm transcription and expression of the majority genes in D. mccartyi genomes. A composite genome of two highly similar D. mccartyi strains (KB-1 Dhc) from the KB-1 metagenome sequence was constructed, and operon prediction was conducted for this composite genome and other single genomes. This operon analysis, together with the quality threshold clustering analysis of transcriptomic data helped generate experimentally testable hypotheses regarding the function of a number of hypothetical proteins and the poorly understood mechanism of energy conservation in D. mccartyi. We also identified functionally enriched important clusters (13 for strain 195 and 11 for KB-1 Dhc) of co-expressed metabolic genes using information from the reconstructed metabolic network. This analysis highlighted some metabolic genes and processes, including lipid metabolism, energy metabolism, and transport that potentially play important roles in organohalide respiration. Overall, this study shows the importance of an organism’s metabolic reconstruction in analyzing various ‘‘omics’’ data to obtain improved understanding of the metabolism and physiology of the organism

Complete genome sequence of Dehalogenimonas lykanthroporepellens type strain (BL-DC-9T) and comparison to “Dehalococcoides” strains

Dehalogenimonas lykanthroporepellens is the type species of the genus Dehalogenimonas, which belongs to a deeply branching lineage within the phylum Chloroflexi. This strictly anaerobic, mesophilic, non spore-forming, Gram-negative staining bacterium was first isolated from chlorinated solvent contaminated groundwater at a Superfund site located near Baton Rouge, Louisiana, USA. D. lykanthroporepellens was of interest for genome sequencing for two reasons: (a) an unusual ability to couple growth with reductive dechlorination of environmentally important polychlorinated aliphatic alkanes and (b) a phylogenetic position that is distant from previously sequenced bacteria. The 1,686,510 bp circular chromosome of strain BL-DC-9T contains 1,720 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small subunit rRNA (16S) locus

Chasing organohalide respirers: ecogenomics approaches to assess the bioremediation capacity of soils

Het opsporen van organohalogeen-reducerende bacteriën: ecogenomics benaderingen om de bioremediatie-capaciteit van de bodem te beoordelen. Organohalogeen-reducerende bacteriën (OHRB) zijn efficiënte afbrekers van organische chloorverbindingen, zoals gechloreerde ethenen, chloorfenolen en andere gehalogeneerde alifatische en aromatische koolwaterstoffen. Desondanks, lijken deze organische chloorverbindingen te volharden op verschillende locaties. De reden voor dit gebrek aan afbraak kan worden toegeschreven aan het ontbreken van OHRB in voldoende aantallen of aan verkeerde fysisch-chemische omstandigheden voor hun groei en activiteit. Derhalve is er een dringende behoefte aan snelle, robuuste en gevoelige methoden die het voorspellen van en het toezicht houden op het bioremediatie potentieel en de activiteit van OHRB mogelijk maken. Moleculaire monitoring en modelsimulaties werden toegepast om de in-situ afbraak prestaties van een on-site dechlorerende bioreactor te bepalen en zijn invloed op de vervuilingsspluim. De toepasbaarheid van dit systeem werd getest in verschillende verontreinigde bodems

Characterizing the metabolism of Dehalococcoides with a constraint-based model

Dehalococcoides strains respire a wide variety of chloro-organic compounds and are important for the bioremediation of toxic, persistent, carcinogenic, and ubiquitous ground water pollutants. In order to better understand metabolism and optimize their application, we have developed a pan-genome-scale metabolic network and constraint-based metabolic model of Dehalococcoides. The pan-genome was constructed from publicly available complete genome sequences of Dehalococcoides sp. strain CBDB1, strain 195, strain BAV1, and strain VS. We found that Dehalococcoides pan-genome consisted of 1118 core genes (shared by all), 457 dispensable genes (shared by some), and 486 unique genes (found in only one genome). The model included 549 metabolic genes that encoded 356 proteins catalyzing 497 gene-associated model reactions. Of these 497 reactions, 477 were associated with core metabolic genes, 18 with dispensable genes, and 2 with unique genes. This study, in addition to analyzing the metabolism of an environmentally important phylogenetic group on a pan-genome scale, provides valuable insights into Dehalococcoides metabolic limitations, low growth yields, and energy conservation. The model also provides a framework to anchor and compare disparate experimental data, as well as to give insights on the physiological impact of "incomplete" pathways, such as the TCA-cycle, CO 2 fixation, and cobalamin biosynthesis pathways. The model, referred to as iAI549, highlights the specialized and highly conserved nature of Dehalococcoides metabolism, and suggests that evolution of Dehalococcoides species is driven by the electron acceptor availability

Syntrophic Partners Enhance Growth and Respiratory Dehalogenation of Hexachlorobenzene by Dehalococcoides mccartyi Strain CBDB1

This study investigated syntrophic interactions between chlorinated benzene respiring Dehalococcoides mccartyi strain CBDB1 and fermenting partners (Desulfovibrio vulgaris, Syntrophobacter fumaroxidans, and Geobacter lovleyi) during hexachlorobenzene respiration. Dechlorination rates in syntrophic co-cultures were enhanced 2-3 fold compared to H2 fed CBDB1 pure cultures (0.23 ± 0.04 μmol Cl− day−1). Syntrophic partners were also able to supply cobalamins to CBDB1, albeit with 3–10 fold lower resultant dechlorination activity compared to cultures receiving exogenous cyanocobalamin. Strain CBDB1 pure cultures accumulated ~1 μmol of carbon monoxide per 87.5 μmol Cl− released during hexachlorobenzene respiration resulting in decreases in dechlorination activity. The syntrophic partners investigated were shown to consume carbon monoxide generated by CBDB1, thus relieving carbon monoxide autotoxicity. Accumulation of lesser chlorinated chlorobenzene congeners (1,3- and 1,4-dichlorobenzene and 1,3,5-trichlorobenzene) also inhibited dechlorination activity and their removal from the headspace through adsorption to granular activated carbon was shown to restore activity. Proteomic analysis revealed co-culturing strain CBDB1 with Geobacter lovleyi upregulated CBDB1 genes associated with reductive dehalogenases, hydrogenases, formate dehydrogenase, and ribosomal proteins. These data provide insight into CBDB1 ecology and inform strategies for application of CBDB1 in ex situ hexachlorobenzene destruction technologies

New dehalococcoides species dechlorinate chloroethenes with unusual metabolic pathways

Ph.DDOCTOR OF PHILOSOPH