Anaerobe Atmungsprozesse in Bakterien der Klasse Dehalococcoidia

The bacterial class Dehalococcoidia, phylum Chloroflexi, encompasses several cultivated strains and many so far uncultivated bacteria, known from molecular detection of their 16S rRNA gene sequences in marine sediments. All cultivated representatives exclusively respire with halogenated compounds as terminal electron acceptor in a process termed organohalide respiration. However, it is not known whether organohalide respiration can explain the abundance and ubiquitous distribution of Dehalococcoidia in marine pristine sediments. In the current work, electron acceptors for respiration of Dehalococcoidia were investigated with the model organism Dehalococcoides mccartyi strain CBDB1 to gain insight into which properties of halogenated compounds are required to serve as electron acceptor of Dehalococcoidia and might in part explain their abundance at marine sites. For this, a microtiter plate-based assay was established to measure reductive dehalogenase activity – the key enzyme in organohalide respiration. D. mccartyi strain CBDB1 dehalogenated chlorinated benzonitriles, chlorinated anilines and brominated phenols, benzenes, pyridines, furoic and benzoic acids and specific activities of 4.5 to 241 nkat mg^-1 protein were determined. In cultivation experiments, growth yields of 0.1 x 10^14 to 2.3 x 10^14 cells mol^-1 halogen released were obtained with strain CBDB1. Brominated electron acceptors were generally dehalogenated to a further extent than their chlorinated equivalents and showed higher specific activity rates in resting cell assays. Results of shotgun proteomics and activity assays suggest that the same reductive dehalogenases are involved in the dehalogenation of brominated and chlorinated benzenes, indicating chemical properties influence reductive dehalogenation patterns of strain CBDB1. The correlation of density functional theory calculations with microbial and biochemical experiments revealed that functional groups decreasing electron density at the halogen and not at the halogen-substituted carbon enhance reductive dehalogenation. The most positive halogen partial charge predicted the regioselective dehalogenation catalysed by strain CBDB1 for up to 96% of all evaluated molecules. These findings suggest a cobalamin–halogen interaction during reductive dehalogenation which stands in contrast to previous models of reductive dehalogenation. A halogen– cobalamin interaction could in part explain the broad electron acceptor diversity of organohaliderespiring Dehalococcoidia suggesting that a multitude of natural organohalogens – including brominated aromatics – could serve as natural halogenated electron acceptor for respiration and growth of Dehalococcoidia in marine sediments. In addition, the established prediction systems may allow for improved fate prediction of halogenated compounds from anthropogenic or natural sources in the environment. Reductive dehalogenation of brominated aromatics was also shown to occur in marine deep-sea sediment microcosms with activity assays, inhibition studies and cultivation experiments although no Dehalococcoidia were detected. However, the conducted experiments demonstrated that similar dehalogenation patterns observed with model organism strain CBDB1 occur in non-contaminated deep-sea sediments. To investigate the potential of Dehalococcoidia to respire non-halogenated electron acceptors, the genetic information from the single amplified Dehalococcoidia genome SAG-C11 was used to design primers for a newly identified class of dissimilatory sulphite reduction genes (dsr) in Chloroflexi. The primers were used to study sediment samples from different locations and depths for the presence of Chloroflexi-related dsr genes. Several dsr genes located in a similar genetic context as observed in SAG-C11 and affiliating with dsr genes from SAG-C11 in phylogenetic analyses were detected in sediments from Aarhus, the Baffin Bay and in tidal flat sediments of the Wadden Sea. The obtained dsrAB genes shared 71–100% nucleotide sequence identities with dsrAB of SAG-C11 suggesting that dissimilatory sulphite reduction is a more widespread mode of respiration in the class Dehalococcoidia and could contribute together with organohalide respiration of brominated natural electron acceptors to the abundance and ubiquitous presence of Dehalococcoidia in marine sediments.Die vorliegende Arbeit beschreibt Formen der anaeroben Atmung der bakteriellen Klasse Dehalococcoidia, Abteilung Chloroflexi, welche zu den häufigsten und abundantesten Bakteriengruppen mariner Sedimente gehört und deren kultivierten Vertreter aus terrestrischen Habitaten ausnahmslos auf eine Atmung mit halogenierten organischen Verbindungen (Organohalidatmung) angewiesen sind. Kultivierung und Mikrotiterplatten-basierte Aktivitätstests mit strukturell unterschiedlichen Elektronenakzeptoren zeigten, dass Dehalococcoidia Modellorganismus Stamm CBDB1 chlorierte Benzonitrile, chlorierte Aniline und bromierte Phenole, Benzole, Pyridine, Furonund Benzoesäuren dehalogenierte. Wachstumsausbeuten von 0,1 x 10^14 bis 2,3 x 10^14 Zellen pro Mol freigesetztem Halogen Anion und spezifische Aktivitäten von 4,5 bis 241 nkat pro mg Protein wurden gemessen. Bromierte Aromaten wurden weitergehend und mit höheren spezifischen Aktivitäten dehalogeniert als ihre chlorierten Äquivalente. Aktivitätstests und Shot-Gun Proteomik deuteten darauf hin, dass die gleichen reduktiven Dehalogenasen in die Umsetzung der getesteten bromierten und chlorierten Benzole involviert sind. Bromierte aromatische Verbindungen, die in marine Habitaten vorkommen, könnten somit als natürliche Elektronenakzeptoren für Organohalid-atmende Dehalococcoidia in marinen Sedimenten dienen. Eine reduktive Dehalogenierung bromierter Verbindungen wurde auch in Aktivitätstests mit Zellen aus marinen Sediment-Mikrokosmen und durch Inhibitionsstudien nachgewiesen. Obwohl keine Chloroflexi in den marinen Sediment- Mikrokosmen detektiert werden konnten, zeigten die Experimente, dass reduktive Dehalogenierung auch in marinen nicht-kontaminierten Tiefseesedimenten vorkommen kann. Die Korrelation mikrobiologischer und biochemischer Versuche mit Dichtefunktionaltheorie-basierten Berechnungen verschiedener Partialladungs-Modelle zeigte, dass die regioselektive Dehalogenierung durch Stamm CBDB1 mit Hilfe der positivsten Halogen-Partialladung für 96% der untersuchten Moleküle vorausgesagt werden konnte. Außerdem konnte gezeigt werden, dass funktionelle Gruppen, die die Elektronendichte am Halogenatom verringern, die reduktive Dehalogenierung unterstützen. Dies weist auf eine Cobalamin–Halogen Interaktion während der reduktiven Dehalogenierung hin und steht im Gegensatz zu bisherigen Modellen der reduktiven Dehalogenierung. Eine Cobalamin–Halogen Interaktion könnte dazu beitragen, das breite Elektronenakzeptoren Spektrum Organohalid-atmender Dehalococcoidia zu erklären und könnte Organohalid-atmenden Dehalococcoidia in marinen Sedimenten erlauben, verschiedenste halogenierte Verbindungen wie z.B. bromierte komplexe Aromaten für die Atmung zu nutzen. In einem weiteren Teil dieser Dissertation wurde mit molekularbiologischen Methoden das Potential von Dehalococcoidia untersucht, nicht-halogenierte Elektronenakzeptoren zu nutzen. Dies könnte die Stratifikation von Dehalococcoidia Subgruppen entlang geochemischer Gradienten erklären. Dazu wurden mit Hilfe der Genominformation einer Dehalococcoidia-Einzelzelle („SAG-C11“) PCR-Primer entwickelt, die spezifisch dsr Gene in Dehalococcoidia amplifizierten. Mit diesen Primern wurden das Vorkommen und die Diversität der dissimilatorischen Sulfitreduktase (dsr) in Dehalococcoidia in Sedimenten verschiedener Orte und Tiefen untersucht. Unterschiedliche dsr Gene, die sich in einem ähnlichen genetischen Kontext wie in SAG-C11 befanden und in phylogenetischen Analysen mit dsr Sequenzen aus SAG-C11 gruppierten, wurden in Sedimenten der Aarhusbucht, der Baffinbucht und in Wattsedimenten der Nordsee nachgewiesen. Nukleotidsequenzähnlichkeiten von 71–100% der aus marinen Sedimenten amplifizierten dsrAB-Sequenzen zu den dsrAB-Sequenzen aus SAG-C11 weisen darauf hin, dass dissimilatorische Sulfitreduktion ein verbreiteter Atmungsprozess in Dehalococcoidia ist und zusammen mit der Organohalidatmung bromierter Verbindungen zu einer Erklärung der ubiquitären Verbreitung von Dehalococcoidia in marine Sedimenten in unterschiedlichen biogeochemischen Zonen beitragen kann.EC/FP7/202903/EU/Microbiology of Dehalococcoides-like Chloroflexi/MICROFLE

Characterization of phage vB_EcoS-EE09 infecting <i>E. coli</i> DSM613 Isolated from Wastewater Treatment Plant Effluent and Comparative Proteomics of the Infected and Non-Infected Host

Phages influence microbial communities, can be applied in phage therapy, or may serve as bioindicators, e.g., in (waste)water management. We here characterized the Escherichia phage vB_EcoS-EE09 isolated from an urban wastewater treatment plant effluent. Phage vB_EcoS-EE09 belongs to the genus Dhillonvirus, class Caudoviricetes. It has an icosahedral capsid with a long non-contractile tail and a dsDNA genome with an approximate size of 44 kb and a 54.6% GC content. Phage vB_EcoS-EE09 infected 12 out of the 17 E. coli strains tested. We identified 16 structural phage proteins, including the major capsid protein, in cell-free lysates by protein mass spectrometry. Comparative proteomics of protein extracts of infected E. coli cells revealed that proteins involved in amino acid and protein metabolism were more abundant in infected compared to non-infected cells. Among the proteins involved in the stress response, 74% were less abundant in the infected cultures compared to the non-infected controls, with six proteins showing significant less abundance. Repressing the expression of these proteins may be a phage strategy to evade host defense mechanisms. Our results contribute to diversifying phage collections, identifying structural proteins to enable better reliability in annotating taxonomically related phage genomes, and understanding phage–host interactions at the protein level

Distinct Carbon Isotope Fractionation Signatures during Biotic and Abiotic Reductive Transformation of Chlordecone

International audienceChlordecone is a synthetic organochlorine pesticide, extensively used in banana plantations of the French West Indies from 1972 to 1993. Due to its environmental persistence and bioaccumulation, it has dramatic public health and socio-economic impact. Here we describe a method for carbon-directed compound specific isotope analysis (CSIA) for chlordecone and apply it to monitor biotic and abiotic reductive transformation reactions, selected on the basis of their distinct product profiles (polychloroindenes versus lower chlorinated hydrochlordecones). Significant carbon isotopic enrichments were observed for all microbially mediated transformations (epsilon(bulk) = -6.8 parts per thousand with a Citrobacter strain and epsilon(bulk) = -4.6 parts per thousand with a bacterial consortium) and for two abiotic transformations (epsilon(bulk) = -4.1 parts per thousand with zerovalent iron and epsilon(bulk) = -2.6 parts per thousand with sodium sulfide and vitamin B-12). The reaction with titanium(III) citrate and vitamin B-12 which shows the product profile most similar to that observed in biotic transformation, led to low carbon isotope enrichment (epsilon(bulk) = -0.8 parts per thousand). The CSIA protocol was also applied on representative chlordecone formulations previously used in the French West Indies, giving similar chlordecone delta C-13 values from -31.1 +/- 0.2 parts per thousand to -34.2 +/- 0.2 parts per thousand for all studied samples. This allows the in situ application of CSIA for the assessment of chlordecone persistence

Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation

Abstract Background Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. Results Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. Conclusions The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water

Anaerobic Microbial Transformation of Halogenated Aromatics and Fate Prediction Using Electron Density Modeling

Halogenated homo- and heterocyclic aromatics including disinfectants, pesticides and pharmaceuticals raise concern as persistent and toxic contaminants with often unknown fate. Remediation strategies and natural attenuation in anaerobic environments often build on microbial reductive dehalogenation. Here we describe the transformation of halogenated anilines, benzonitriles, phenols, methoxylated, or hydroxylated benzoic acids, pyridines, thiophenes, furoic acids, and benzenes by <i>Dehalococcoides mccartyi</i> strain CBDB1 and environmental fate modeling of the dehalogenation pathways. The compounds were chosen based on structural considerations to investigate the influence of functional groups present in a multitude of commercially used halogenated aromatics. Experimentally obtained growth yields were 0.1 to 5 × 10¹⁴ cells mol^–1 of halogen released (corresponding to 0.3–15.3 g protein mol^–1 halogen), and specific enzyme activities ranged from 4.5 to 87.4 nkat mg^–1 protein. Chlorinated electron-poor pyridines were not dechlorinated in contrast to electron-rich thiophenes. Three different partial charge models demonstrated that the regioselective removal of halogens is governed by the least negative partial charge of the halogen. Microbial reaction pathways combined with computational chemistry and pertinent literature findings on Co^I chemistry suggest that halide expulsion during reductive dehalogenation is initiated through single electron transfer from B₁₂Co^I to the apical halogen site

Single-Cell Genome and Group-Specific dsrAB Sequencing Implicate Marine Members of the Class Dehalococcoidia (Phylum Chloroflexi) in Sulfur Cycling

The marine subsurface sediment biosphere is widely inhabited by bacteria affiliated with the class Dehalococcoidia (DEH), phylum Chloroflexi, and yet little is known regarding their metabolisms. In this report, genomic content from a single DEH cell (DEH-C11) with a 16S rRNA gene that was affiliated with a diverse cluster of 16S rRNA gene sequences prevalent in marine sediments was obtained from sediments of Aarhus Bay, Denmark. The distinctive gene content of this cell suggests metabolic characteristics that differ from those of known DEH and Chloroflexi. The presence of genes encoding dissimilatory sulfite reductase (Dsr) suggests that DEH could respire oxidized sulfur compounds, although Chloroflexi have never been implicated in this mode of sulfur cycling. Using long-range PCR assays targeting DEH dsr loci, dsrAB genes were amplified and sequenced from various marine sediments. Many of the amplified dsrAB sequences were affiliated with the DEH Dsr clade, which we propose equates to a family-level clade. This provides supporting evidence for the potential for sulfite reduction by diverse DEH species. DEH-C11 also harbored genes encoding reductases for arsenate, dimethyl sulfoxide, and halogenated organics. The reductive dehalogenase homolog (RdhA) forms a monophyletic clade along with RdhA sequences from various DEH-derived contigs retrieved from available metagenomes. Multiple facts indicate that this RdhA may not be a terminal reductase. The presence of other genes indicated that nutrients and energy may be derived from the oxidation of substituted homocyclic and heterocyclic aromatic compounds. Together, these results suggest that marine DEH play a previously unrecognized role in sulfur cycling and reveal the potential for expanded catabolic and respiratory functions among subsurface DEH

Single-Cell Genome and Group-SpecificdsrABSequencing Implicate Marine Members of the ClassDehalococcoidia(PhylumChloroflexi) in Sulfur Cycling

The marine subsurface sediment biosphere is widely inhabited by bacteria affiliated with the class Dehalococcoidia (DEH), phylum Chloroflexi, and yet little is known regarding their metabolisms. In this report, genomic content from a single DEH cell (DEH-C11) with a 16S rRNA gene that was affiliated with a diverse cluster of 16S rRNA gene sequences prevalent in marine sediments was obtained from sediments of Aarhus Bay, Denmark. The distinctive gene content of this cell suggests metabolic characteristics that differ from those of known DEH and Chloroflexi. The presence of genes encoding dissimilatory sulfite reductase (Dsr) suggests that DEH could respire oxidized sulfur compounds, although Chloroflexi have never been implicated in this mode of sulfur cycling. Using long-range PCR assays targeting DEH dsr loci, dsrAB genes were amplified and sequenced from various marine sediments. Many of the amplified dsrAB sequences were affiliated with the DEH Dsr clade, which we propose equates to a family-level clade. This provides supporting evidence for the potential for sulfite reduction by diverse DEH species. DEH-C11 also harbored genes encoding reductases for arsenate, dimethyl sulfoxide, and halogenated organics. The reductive dehalogenase homolog (RdhA) forms a monophyletic clade along with RdhA sequences from various DEH-derived contigs retrieved from available metagenomes. Multiple facts indicate that this RdhA may not be a terminal reductase. The presence of other genes indicated that nutrients and energy may be derived from the oxidation of substituted homocyclic and heterocyclic aromatic compounds. Together, these results suggest that marine DEH play a previously unrecognized role in sulfur cycling and reveal the potential for expanded catabolic and respiratory functions among subsurface DEH. IMPORTANCE: Sediments underlying our oceans are inhabited by microorganisms in cell numbers similar to those estimated to inhabit the oceans. Microorganisms in sediments consist of various diverse and uncharacterized groups that contribute substantially to global biogeochemical cycles. Since most subsurface microorganisms continue to evade cultivation, possibly due to very slow growth, we obtained and analyzed genomic information from a representative of one of the most widespread and abundant, yet uncharacterized bacterial groups of the marine subsurface. We describe several key features that may contribute to their widespread distribution, such as respiratory flexibility and the potential to use oxidized sulfur compounds, which are abundant in marine environments, as electron acceptors. Together, these data provide important information that can be used to assist in designing enrichment strategies or other postgenomic studies, while also improving our understanding of the diversity and distribution of dsrAB genes, which are widely used functional marker genes for sulfur-cycling microbes