An in vitro reconstitution system to monitor iron transfer to the active site during the maturation of [NiFe]-hydrogenase.

[NiFe]-hydrogenases (Hyds) comprise a small and a large subunit. The latter harbors the biologically unique [NiFe](CN)2CO active-site cofactor. The maturation process includes the assembly of the [Fe](CN)2CO cofactor precursor, nickel binding, endoproteolytic cleavage of the large subunit, and dimerization with the small subunit to yield active enzyme. The biosynthesis of the [Fe](CN)2CO moiety of [NiFe]-Hyd-1 and Hyd-2 occurs on the scaffold complex HybG-HypD (GD), whereas the HypC-HypD complex is specific for the assembly of Hyd-3. The metabolic source and the route for delivering iron to the active site remain unclear. To investigate the maturation process of O2-tolerant Hyd-1 from Escherichia coli, we developed an enzymatic in vitro reconstitution system that allows for the synthesis of Hyd-1 using only purified components. Together with this in vitro reconstitution system, we employed biochemical analyses, infrared spectroscopy (attenuated total reflection FTIR), mass spectrometry (MS), and microscale thermophoresis to monitor the iron transfer during the maturation process and to understand how the [Fe](CN)2CO cofactor precursor is ultimately incorporated into the large subunit. We demonstrate the direct transfer of iron from 57Fe-labeled GD complex to the large subunit of Hyd-1. Our data reveal that the GD complex exclusively interacts with the large subunit of Hyd-1 and Hyd-2 but not with the large subunit of Hyd-3. Furthermore, we show that the presence of iron in the active site is a prerequisite for nickel insertion. Taken together, these findings reveal how the [Fe](CN)2CO cofactor precursor is transferred and incorporated into the active site of [NiFe]-Hyd.

Respiratory protein interactions in Dehalobacter sp. strain 8M revealed through genomic and native proteomic analyses.

Dehalobacter (Firmicutes) encompass obligate organohalide-respiring bacteria used for bioremediation of groundwater contaminated with halogenated organics. Various aspects of their biochemistry remain unknown, including the identities and interactions of respiratory proteins. Here, we sequenced the genome of Dehalobacter sp. strain 8M and analysed its protein expression. Strain 8M encodes 22 reductive dehalogenase homologous (RdhA) proteins. RdhA D8M_v2_40029 (TmrA) was among the two most abundant proteins during growth with trichloromethane and 1,1,2-trichloroethane. To examine interactions of respiratory proteins, we used blue native gel electrophoresis together with dehalogenation activity tests and mass spectrometry. The highest activities were found in gel slices with the highest abundance of TmrA. Protein distributions across gel lanes provided biochemical evidence that the large and small subunits of the membrane-bound [NiFe] uptake hydrogenase (HupL and HupS) interacted strongly and that HupL/S interacted weakly with RdhA. Moreover, the interaction of RdhB and membrane-bound b-type cytochrome HupC was detected. RdhC proteins, often encoded in rdh operons but without described function, migrated in a protein complex not associated with HupL/S or RdhA. This study provides the first biochemical evidence of respiratory protein interactions in Dehalobacter, discusses implications for the respiratory architecture and advances the molecular comprehension of this unique respiratory chain.

Insight into the Mechanism Underlying Dehalococcoides mccartyi Strain CBDB1-Mediated B12-Dependent Aromatic Reductive Dehalogenation.

Anaerobic bacteria transform aromatic halides through reductive dehalogenation. This dehalorespiration is catalyzed by the supernucleophilic coenzyme vitamin B12, cob(I)alamin, in reductive dehalogenases. So far, the underlying inner-sphere electron transfer (ET) mechanism has been discussed controversially. In the present study, all 36 chloro-, bromo-, and fluorobenzenes and full-size cobalamin are analyzed at the quantum chemical density functional theory level with respect to a wide range of theoretically possible inner-sphere ET mechanisms. The calculated reaction free energies within the framework of CoI···X (X = F, Cl, and Br) attack rule out most of the inner-sphere pathways. The only route with feasible energetics is a proton-coupled two-ET mechanism that involves a B12 side-chain tyrosine (modeled by phenol) as a proton donor. For 12 chlorobenzenes and 9 bromobenzenes with experimental data from Dehalococcoides mccartyi strain CBDB1, the newly proposed PC-TET mechanism successfully discriminates 16 of 17 active from 4 inactive substrates and correctly predicts the observed regiospecificity to 100%. Moreover, fluorobenzenes are predicted to be recalcitrant in agreement with experimental findings. Conceptually, based on the Bell-Evans-Polanyi principle, the computational approach provides novel mechanistic insights and may serve as a tool for predicting the energetic feasibility of reductive aromatic dehalogenation.

Improve Niche Colonization and Microbial Interactions for Organohalide-Respiring-Bacteria-Mediated Remediation of Chloroethene-Contaminated Sites.

Organohalide-respiring bacteria (OHRB)-mediated reductive dehalogenation is promising in in situ bioremediation of chloroethene-contaminated sites. The bioremediation efficiency of this approach is largely determined by the successful colonization of fastidious OHRB, which is highly dependent on the presence of proper growth niches and microbial interactions. In this study, based on two ecological principles (i.e., Priority Effects and Coexistence Theory), three strategies were developed to enhance niche colonization of OHRB, which were tested both in laboratory experiments and field applications: (i) preinoculation of a niche-preparing culture (NPC, being mainly constituted of fermenting bacteria and methanogens); (ii) staggered fermentation; and (iii) increased inoculation of CE40 (a Dehalococcoides-containing tetrachloroethene-to-ethene dechlorinating enrichment culture). Batch experimental results show significantly higher dechlorination efficiencies, as well as lower concentrations of volatile fatty acids (VFAs) and methane, in experimental sets with staggered fermentation and niche-preconditioning with NPC for 4 days (CE40_NPC-4) relative to control sets. Accordingly, a comparatively higher abundance of Dehalococcoides as major OHRB, together with a lower abundance of fermenting bacteria and methanogens, was observed in CE40_NPC-4 with staggered fermentation, which indicated the balanced syntrophic and competitive interactions between OHRB and other populations for the efficient dechlorination. Further experiments with microbial source tracking analyses suggested enhanced colonization of OHRB by increasing the inoculation ratio of CE40. The optimized conditions for enhanced colonization of OHRB were successfully employed for field bioremediation of trichloroethene (TCE, 0.3-1.4 mM)- and vinyl chloride (VC, ∼0.04 mM)-contaminated sites, resulting in 96.6% TCE and 99.7% VC dechlorination to ethene within 5 and 3 months, respectively. This study provides ecological principles-guided strategies for efficient bioremediation of chloroethene-contaminated sites, which may be also employed for removal of other emerging organohalide pollutants.

Mechanistic insight into the Dehalococcoides-mediated reductive dechlorination of polychlorinated biphenyls.

Microbial reductive dechlorination provides a green and highly desirable approach to address the pollution raised by the substantial legacies of polychlorinated biphenyls (PCBs) in soil, sediment, and underground water. It has been shown that the reaction event is catalyzed by supernucleophilic cob(I)alamin housed in reductive dehalogenases (RDases). However, the mechanism still remains elusive. Herein, we unravel the mechanism via quantum chemical calculations, considering a general model of RDase and the dechlorination regioselectivity of two representative PCB congeners, 234-236-CB and 2345-236-CB. The B12-catalzyed reductive dechlorination of PCBs starts with the formation of a reactant complex, followed by a proton-coupled two-electron transfer (PC-TET) and a subsequent single-electron transfer (SET). The PC-TET yields a cob(III)alamin-featured intermediate, which is quickly reduced by the latter SET fueled by significant energetic benefits (∼100 kcal mol-1). It rationalizes the exclusive detection and characterization of cob(I/II)alamins in RDase-mediated dehalogenation experiments. The determined mechanism successfully reproduces the experimental dechlorination regioselectivity and reactivity, as observed with Dehalococcoides mccartyi strain CG1.

Effects of reducing, stabilizing, and antibiotic agents on "Candidatus Kuenenia stuttgartiensis".

Anaerobic ammon ium oxidizing (anammox) bacteria oxidize ammonium and reduce nitrite, producing N2, and could play a major role in energy-optimized wastewater treatment. However, sensitivity to various environmental conditions and slow growth currently hinder their wide application. Here, we attempted to determine online the effect of environmental stresses on anammox bacteria by using an overnight batch activity test with whole cells, in which anammox activity was calculated by quantifying N2 production via headspace-pressure monitoring. A planktonic mixed culture dominated by "Candidatus Kuenenia stuttgartiensis" strain CSTR1 was cultivated in a 30-L semi-continuous stirring tank reactor. In overnight resting-cell anammox activity tests, oxygen caused strong inhibition of anammox activity, which was reversed by sodium sulfite (30 µM). The tested antibiotics sulfamethoxazole, kanamycin, and ciprofloxacin elicited their effect on a dose-dependent manner; however, strain CSTR1 was highly resistant to sulfamethoxazole. Anammox activity was improved by activated carbon and Fe2O3. Protein expression analysis from resting cells after anammox activity stimulation revealed that NapC/NirT family cytochrome c (KsCSTR_12840), hydrazine synthase, hydrazine dehydrogenase, hydroxylamine oxidase, and nitrate:nitrite oxidoreductase were upregulated, while a putative hydroxylamine oxidoreductase HAO (KsCSTR_49490) was downregulated. These findings contribute to the growing knowledge on anammox bacteria physiology, eventually leading to the control of anammox bacteria growth and activity in real-world application. KEY POINTS: • Sulfite additions can reverse oxygen inhibition of the anammox process • Anammox activity was improved by activated carbon and ferric oxide • Sulfamethoxazole marginally affected anammox activity.

GroEL-Proteotyping of Bacterial Communities Using Tandem Mass Spectrometry.

Profiling bacterial populations in mixed communities is a common task in microbiology. Sequencing of 16S small subunit ribosomal-RNA (16S rRNA) gene amplicons is a widely accepted and functional approach but relies on amplification primers and cannot quantify isotope incorporation. Tandem mass spectrometry proteotyping is an effective alternative for taxonomically profiling microorganisms. We suggest that targeted proteotyping approaches can complement traditional population analyses. Therefore, we describe an approach to assess bacterial community compositions at the family level using the taxonomic marker protein GroEL, which is ubiquitously found in bacteria, except a few obligate intracellular species. We refer to our method as GroEL-proteotyping. GroEL-proteotyping is based on high-resolution tandem mass spectrometry of GroEL peptides and identification of GroEL-derived taxa via a Galaxy workflow and a subsequent Python-based analysis script. Its advantage is that it can be performed with a curated and extendable sample-independent database and that GroEL can be pre-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to reduce sample complexity, improving GroEL identification while simultaneously decreasing the instrument time. GroEL-proteotyping was validated by employing it on a comprehensive raw dataset obtained through a metaproteome approach from synthetic microbial communities as well as real human gut samples. Our data show that GroEL-proteotyping enables fast and straightforward profiling of highly abundant taxa in bacterial communities at reasonable taxonomic resolution.

Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazole.

Thiabendazole (TBZ), is a persistent fungicide/anthelminthic and a serious environmental threat. We previously enriched a TBZ-degrading bacterial consortium and provided first evidence for a Sphingomonas involvement in TBZ transformation. Here, using a multi-omic approach combined with DNA-stable isotope probing (SIP) we verified the key degrading role of Sphingomonas and identify potential microbial interactions governing consortium functioning. SIP and amplicon sequencing analysis of the heavy and light DNA fraction of cultures grown on 13 C-labelled versus 12 C-TBZ showed that 66% of the 13 C-labelled TBZ was assimilated by Sphingomonas. Metagenomic analysis retrieved 18 metagenome-assembled genomes with the dominant belonging to Sphingomonas, Sinobacteriaceae, Bradyrhizobium, Filimonas and Hydrogenophaga. Meta-transcriptomics/-proteomics and non-target mass spectrometry suggested TBZ transformation by Sphingomonas via initial cleavage by a carbazole dioxygenase (car) to thiazole-4-carboxamidine (terminal compound) and catechol or a cleaved benzyl ring derivative, further transformed through an ortho-cleavage (cat) pathway. Microbial co-occurrence and gene expression networks suggested strong interactions between Sphingomonas and a Hydrogenophaga. The latter activated its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway to satisfy its B12 auxotrophy. Our findings indicate microbial interactions aligning with the 'black queen hypothesis' where Sphingomonas (detoxifier, B12 recipient) and Hydrogenophaga (B12 producer, enjoying detoxification) act as both helpers and beneficiaries.

Structure and functional capacity of a benzene-mineralizing, nitrate-reducing microbial community.

AIMS: How benzene is metabolized by microbes under anoxic conditions is not fully understood. Here, we studied the degradation pathways in a benzene-mineralizing, nitrate-reducing enrichment culture. METHODS AND RESULTS: Benzene mineralization was dependent on the presence of nitrate and correlated to the enrichment of a Peptococcaceae phylotype only distantly related to known anaerobic benzene degraders of this family. Its relative abundance decreased after benzene mineralization had terminated, while other abundant taxa-Ignavibacteriaceae, Rhodanobacteraceae and Brocadiaceae-slightly increased. Generally, the microbial community remained diverse despite the amendment of benzene as single organic carbon source, suggesting complex trophic interactions between different functional groups. A subunit of the putative anaerobic benzene carboxylase previously detected in Peptococcaceae was identified by metaproteomic analysis suggesting that benzene was activated by carboxylation. Detection of proteins involved in anaerobic ammonium oxidation (anammox) indicates that benzene mineralization was accompanied by anammox, facilitated by nitrite accumulation and the presence of ammonium in the growth medium. CONCLUSIONS: The results suggest that benzene was activated by carboxylation and further assimilated by a novel Peptococcaceae phylotype. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm the hypothesis that Peptococcaceae are important anaerobic benzene degraders.

Thermophilic archaea activate butane via alkyl-coenzyme M formation.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding ß-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly.

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.

Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M.

Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146-242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron-sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone-independent respiration in Dehalogenimonas.

An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone.

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.

Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase.

Molecular hydrogen (H2) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H2 production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H2 syntheses rely mainly on light, water, and CO2 and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H2-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O2) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O2-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O2-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O2 sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O2 tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH+ to utilize H2 as sole electron source to fix CO2. Syn_ReSH+ also was able to produce H2 under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H2 production.

Extracellular and Intracellular Lanthanide Accumulation in the Methylotrophic Beijerinckiaceae Bacterium RH AL1.

Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in the Beijerinckiaceae bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species, with a preference for heavier lanthanides. Its methanol oxidation machinery is supposedly adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on transcriptome sequencing (RNA-seq) analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the lut cluster but potentially also a type VI secretion system. A high level of constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. IMPORTANCE Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work with commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. The Beijerinckiaceae bacterium RH AL1 might be an attractive target for developing biorecovery strategies to obtain these economically highly demanded metals in environmentally friendly ways.

Anaerobic Transformation and Detoxification of Sulfamethoxazole by Sulfate-Reducing Enrichments and Desulfovibrio vulgaris.

Sulfamethoxazole (SMX) is a veterinary antibiotic that is not efficiently removed from wastewater by routine treatment and therefore can be detected widely in the environment. Here, we investigated whether microbial anaerobic transformation can contribute to the removal of SMX in constructed systems. We enriched SMX-transforming mixed cultures from sediment of a constructed wetland and from digester sludge of a wastewater treatment plant. Transformation of SMX was observed in both sulfate-reducing and methanogenic cultures, whereas nitrate-reducing cultures showed no SMX transformation. In sulfate-reducing cultures, up to 90% of an initial SMX concentration of 100-250 µM was removed within 6 weeks of incubation, and the experiments demonstrated that the transformation was microbially catalyzed. The transformation products in sulfate-reducing cultures were identified as the reduced and isomerized forms of the isoxazole SMX moiety. The transformation products did not spontaneously reoxidize to SMX after oxygen exposure, and their antibacterial activity was significantly decreased compared to SMX. Population analyses in sequential transfers of the sulfate-reducing cultures revealed a community shift toward the genus Desulfovibrio. We therefore tested a deposited strain of Desulfovibrio vulgaris Hildenborough for its capacity to transform SMX and observed the same transformation products and similar transformation rates as in the enrichment cultures. Our work suggests that an initial anaerobic step in wastewater treatment can reduce the concentration of SMX in effluents and could contribute to decreased SMX concentrations in the environment.

Outer-sphere electron transfer does not underpin B12-dependent olefinic reductive dehalogenation in anaerobes.

Anaerobic microbial B12-dependent reductive dehalogenation may pave a way to remediate soil, sediment, and underground water contaminated with halogenated olefins. The chemical reaction is initiated by electron transfer (ET) from supernucleophilic cob(I)alamin (B12s). However, the inherent mechanism as outer-sphere or inner-sphere route is still under debate. To clarify the possibility of an outer-sphere pathway, we calculated free energy barriers of the initial steps of all outer-sphere ET routes by Marcus theory employing density functional theory (DFT). For 18 fluorinated, chlorinated, and brominated ethenes as representative olefins, 164 of 165 reactions with free energy barriers larger than 20 kcal mol-1 are not feasible under physiological dehalogenase conditions. Moreover, electronic structure analysis of perbromoethene with an outer-sphere free energy barrier of 18.2 kcal mol-1 reveals no ET initiation down to Co⋯Br and Co⋯C distances of 3.15 Å. The results demonstrate that the B12-catalyzed reductive dechlorination of olefins in microbes should proceed through an inner-sphere ET pathway.

Comparative genomics in "Candidatus Kuenenia stuttgartiensis" reveal high genomic plasticity in the overall genome structure, CRISPR loci and surface proteins.

BACKGROUND: Anaerobic ammonium oxidizing bacteria (anammox bacteria) are contributing significantly to the nitrogen cycle and are successfully used in wastewater treatment. Due to the lack of complete genomes in the databases, little is known about the stability and variability of their genomes and how the genomes evolve in response to changing environments. RESULTS: Here we report the complete genome of the anammox bacterium "Candidatus Kuenenia stuttgartiensis" strain CSTR1 which was enriched planktonically in a semi-continuous stirred-tank reactor. A comparison of the genome of strain CSTR1 with the genome of "Ca. Kuenenia stuttgartiensis" MBR1 and the draft genome of KUST showed > 99% average nucleotide identity among all. Rearrangements of large genomic regions were observed, most of which were associated with transposase genes. Phylogenetic analysis suggests that strain MBR1 is more distantly related to the other two strains. Proteomic analysis of actively growing cells of strain CSTR1 (growth rate ~ 0.33 d- 1) failed to detect the annotated cytochrome cd1-type nitrite reductase (NirS) although in total 1189 proteins were found in the proteome. Yet, this NirS was expressed when strain CSTR1 was under stress or starvation (growth rate < 0.06 d- 1). We also observed large sequence shifts in the strongly expressed S-layer protein compared to other "Ca. Kuenenia" strains, indicating the formation of hybrids of genes encoding the surface proteins. CONCLUSIONS: "Ca. Kuenenia" strains appear to be relatively stable in their basic physiological traits, but show high variability in overall genome structure and surface proteins.

Transcriptomic and Proteomic Responses of the Organohalide-Respiring Bacterium Desulfoluna spongiiphila to Growth with 2,6-Dibromophenol as the Electron Acceptor.

Organohalide respiration is an important process in the global halogen cycle and for bioremediation. In this study, we compared the global transcriptomic and proteomic analyses of Desulfoluna spongiiphila strain AA1, an organohalide-respiring member of the Desulfobacterota isolated from a marine sponge, with 2,6-dibromophenol or with sulfate as an electron acceptor. The most significant difference of the transcriptomic analysis was the expression of one reductive dehalogenase gene cluster (rdh16), which was significantly upregulated with the addition of 2,6-dibromophenol. The corresponding protein, reductive dehalogenase RdhA16032, was detected in the proteome under treatment with 2,6-dibromophenol but not with sulfate only. There was no significant difference in corrinoid biosynthesis gene expression levels between the two treatments, indicating that the production of corrinoid in D. spongiiphila is constitutive or not specific for organohalide versus sulfate respiration. Electron-transporting proteins or mediators unique for reductive dehalogenation were not revealed in our analysis, and we hypothesize that reductive dehalogenation may share an electron-transporting system with sulfate reduction. The metabolism of D. spongiiphila, predicted from transcriptomic and proteomic results, demonstrates high metabolic versatility and provides insights into the survival strategies of a marine sponge symbiont in an environment rich in organohalide compounds and other secondary metabolites.IMPORTANCE Respiratory reductive dehalogenation is an important process in the overall cycling of both anthropogenic and natural organohalide compounds. Marine sponges produce a vast array of bioactive compounds as secondary metabolites, including diverse halogenated compounds that may enrich for dehalogenating bacteria. Desulfoluna spongiiphila strain AA1 was originally enriched and isolated from the marine sponge Aplysina aerophoba and can grow with both brominated compounds and sulfate as electron acceptors for respiration. An understanding of the overall gene expression and the protein production profile in response to organohalides is needed to identify the full complement of genes or enzymes involved in organohalide respiration. Elucidating the metabolic capacity of this sponge-associated bacterium lays the foundation for understanding how dehalogenating bacteria may control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle.

Dehalococcoides-Mediated B12-Dependent Reductive Dehalogenation of Aromatics Does Not Proceed through Outer-Sphere Electron Transfer.

Several anaerobic bacteria can couple the reduction of aromatic halides to energy conservation. This organohalide respiration is catalyzed by enzymes containing cob(I)alamin, an activated supernucleophilic form of the coenzyme vitamin B12. However, the mechanism underlying the electron transfer (inner-sphere vs outer-sphere ET) still remains elusive. To clarify this issue, we selected 36 fluoro-, chloro-, and bromobenzenes as representative substrates and calculated their free-energy barriers at the quantum chemical density functional theory level, considering a wide range of theoretically possible outer-sphere ET mechanisms. Across all 336 reaction routes addressed, 334 routes involve free-energy barriers larger than 20 kcal/mol. For two reaction routes with highly brominated benzenes, free-energy barriers below 20 kcal/mol imply abiotic reduction as observed in experiments. Thus, microbial B12-dependent aromatic reductive dehalogenation does not proceed through an outer-sphere ET mechanism. Instead, the present study strongly suggests that microbe-catalyzed reductive dehalogenation of aromatic halides is governed by inner-sphere ET.