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.

Characterization of phage vB_EcoS-EE09 infecting E. coli 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.

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.

Yeast gene KTI13 (alias DPH8) operates in the initiation step of diphthamide synthesis on elongation factor 2.

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the DPH1-DPH7 network and an ill-defined KTI13 function. On further examining the latter gene in yeast, we found that kti13Δ null-mutants maintain unmodified EF2 able to escape ADP-ribosylation by DT and to survive EF2 inhibition by sordarin, a diphthamide-dependent antifungal. Consistently, mass spectrometry shows kti13Δ cells are blocked in proper formation of amino-carboxyl-propyl-EF2, the first diphthamide pathway intermediate. Thus, apart from their common function in tRNA modification, both Kti11/Dph3 and Kti13 share roles in the initiation step of EF2 modification. We suggest an alias KTI13/DPH8 nomenclature indicating dual-functionality analogous to KTI11/DPH3.

An Efficient and Scalable Method for the Production of Immunogenic SARS-CoV-2 Virus-like Particles (VLP) from a Mammalian Suspension Cell Line.

The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.

Recently evolved combination of unique sulfatase and amidase genes enables bacterial degradation of the wastewater micropollutant acesulfame worldwide.

Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and an acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA amidase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and likely evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics.

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.

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.

Spatial organization and proteome of a dual-species cyanobacterial biofilm alter among N2-fixing and non-fixing conditions.

Many disciplines have become increasingly interested in cyanobacteria, due to their ability to fix CO2 while using water and sunlight as electron and energy sources. Further, several species of cyanobacteria are also capable of fixing molecular nitrogen, making them independent of the addition of nitrate or ammonia. Thereby they hold huge potential as sustainable biocatalysts. Here, we look into a dual-species biofilm consisting of filamentous diazotrophic cyanobacteria Tolypothrix sp. PCC 7712 and heterotrophic bacteria Pseudomonas taiwanensis VLB 120 growing in a capillary biofilm reactor. Such systems have been reported to enable high cell densities continuous process operation. By combining confocal laser scanning and helium-ion microscopy with a proteomics approach, we examined these organisms' interactions under two nitrogen-feeding strategies: N2-fixing and nitrate assimilation. Not only did Pseudomonas facilitate the biofilm formation by forming a carpet layer on the surface area but also did N2-fixing biofilms show greater attachment to the surface. Pseudomonas proteins related to surface and cell attachments were observed in N2-fixing biofilms in particular. Furthermore, co-localized biofilm cells displayed a resilient response to extra shear forces induced by segmented media/air flows. This study highlights the role of Pseudomonas in the initial attachment process, as well as the effects of different nitrogen-feeding strategies and operation regimes on biofilm composition and growth. IMPORTANCE Cyanobacteria are highly interesting microorganisms due to their ability to synthesize sugars from CO2 while using water and sunlight as electron and energy sources. Further, many species are also capable of utilizing molecular nitrogen, making them independent of artificial fertilizers. In this study, such organisms are cultivated in a technical system, which enables them to attach to the reactor surface, and form three-dimensional structures termed biofilms. Biofilms achieve extraordinarily high cell densities. Furthermore, this growth format allows for continuous processing, both being essential features in biotechnological process development. Understanding biofilm growth and the influence technical settings and media composition have on biofilm maturation and stability are crucial for reaction and reactor design. These findings will help to open up these fascinating organisms for applications as sustainable, resource-efficient industrial workhorses.

Proteogenomics of the novel Dehalobacterium formicoaceticum strain EZ94 highlights a key role of methyltransferases during anaerobic dichloromethane degradation.

Dichloromethane (DCM, methylene chloride) is a toxic, high-volume industrial pollutant of long-standing. Anaerobic biodegradation is crucial for its removal from contaminated environments, yet prevailing mechanisms remain unresolved, especially concerning dehalogenation. In this study, we obtained an assembled genome of a novel DCM-degrading strain, Dehalobacterium formicoaceticum strain EZ94, from a stable DCM-degrading consortium, and we analyzed its proteome during degradation of DCM. A gene cluster recently predicted to play a major role in anaerobic DCM catabolism (the mec cassette) was found. Methyltransferases and other proteins encoded by the mec cassette were among the most abundant proteins produced, suggesting their involvement in DCM catabolism. Reductive dehalogenases were not detected. Genes and corresponding proteins for a complete Wood-Ljungdahl pathway, which could enable further metabolism of DCM carbon, were also found. Unlike for the anaerobic DCM degrader "Ca. F. warabiya," no genes for metabolism of the quaternary amines choline and glycine betaine were identified. This work provides independent and supporting evidence that mec-associated methyltransferases are key to anaerobic DCM metabolism.

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.

Continuous cultivation of Dehalococcoides mccartyi with brominated tyrosine avoids toxic byproducts and gives tight reactor control.

Dehalococcoides mccartyi strain CBDB1 is a strictly anaerobic organohalide-respiring bacterium with strong application potential to remediate aquifers and soils contaminated with halogenated aromatics. To date, cultivation of strain CBDB1 has mostly been done in bottles or fed-batch reactors. Challenges with such systems include low biomass yield and difficulties in controlling the growth conditions. Here, we report the cultivation of planktonic D. mccartyi strain CBDB1 in a continuous stirring tank reactor (CSTR) that led to high cell densities (∼8 × 108 cells mL-1) and dominance of strain CBDB1. The reactor culture received acetate, hydrogen, and the brominated amino acid D- or L-3,5-dibromotyrosine as substrates. Both D- and L-3,5-dibromotyrosine were utilized as respiratory electron acceptors and are promising for biomass production due to their decent solubility in water and the formation of a non-toxic debromination product, tyrosine. By monitoring headspace pressure decrease which is indicative of hydrogen consumption, the organohalide respiration rate was followed in real time. Proteomics analyses revealed that the reductive dehalogenase CbdbA238 was highly expressed with both D- and L-3,5-dibromotyrosine, while other reductive dehalogenases including those that were previously suggested to be constitutively expressed, were repressed. Denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes indicated that the majority of cells in the community belonged to the Dehalococcoides although the CSTR was operated under non-sterile conditions. Hence, tightly controlled CSTR cultivation of Dehalococcoides opens novel options to improve biomass production for bioaugmentation and for advanced biochemical studies.

Carbon and hydrogen stable isotope fractionation of sulfamethoxazole during anaerobic transformation catalyzed by Desulfovibrio vulgaris Hildenborough.

The fate of antibiotics in aquatic environments is of high concern and approaches are needed to assess the transformation of antibiotics in wastewater treatment plants. Here we used the model organism Desulfovibrio vulgaris Hildenborough to analyze compound specific isotope fractionation associated with anaerobic transformation of the antibiotic sulfamethoxazole (SMX). The results show that the rearrangement of the isoxazole ring in SMX is leading to significant carbon and hydrogen isotopic fractionation (εC = -5.8 ± 0.7‰, εH = -34 ± 9‰) during anaerobic transformation. The observed carbon isotopic fractionation is significantly higher than the values reported for aerobic degradation (εC = -0.6 ± 0.1‰) or abiotic reactions (εC = -0.8 to -4.8‰ for photolysis, εC = -0.8 to -2.2‰ for advanced oxidation). This indicates that carbon isotope fractionation can be used as a parameter to differentiate reaction mechanisms of SMX transformation. The corresponding apparent kinetic isotope effect (AKIEC) for anaerobic transformation of SMX was 1.029 ± 0.003, suggesting that the mechanism for anaerobic transformation is distinct from the mechanism reported for microbial aerobic degradation (AKIEC = 1.006 ± 0.001). In addition, dual-element (C-H) isotope analysis of SMX was performed in the present study, which was achieved by utilizing gas chromatography (GC) as the separation method instead of routine liquid chromatography. This dual-element isotope analysis resulted in a Λ value of 4.5 ± 2.2. Overall, compound specific isotope analysis can be a feasible tool to monitor the mitigation of SMX in wastewater treatment plants.

Dehalococcoides mccartyi strain CBDB1 takes up protons from the cytoplasm to reductively dehalogenate organohalides indicating a new modus of proton motive force generation.

Proton translocation across the cytoplasmic membrane is a vital process for all organisms. Dehalococcoides strains are strictly anaerobic organohalide respiring bacteria that lack quinones and cytochromes but express a large membrane-bound protein complex (OHR complex) proposed to generate a proton gradient. However, its functioning is unclear. By using a dehalogenase-based enzyme activity assay with deuterium-labelled water in various experimental designs, we obtained evidence that the halogen atom of the halogenated electron acceptor is substituted with a proton from the cytoplasm. This suggests that the protein complex couples exergonic electron flux through the periplasmic subunits of the OHR complex to the endergonic transport of protons from the cytoplasm across the cytoplasmic membrane against the proton gradient to the halogenated electron acceptor. Using computational tools, we located two proton-conducting half-channels in the AlphaFold2-predicted structure of the OmeB subunit of the OHR complex, converging in a highly conserved arginine residue that could play a proton gatekeeper role. The cytoplasmic proton half-channel in OmeB is connected to a putative proton-conducting path within the reductive dehalogenase subunit. Our results indicate that the reductive dehalogenase and its halogenated substrate serve as both electron and proton acceptors, providing insights into the proton translocation mechanism within the OHR complex and contributing to a better understanding of energy conservation in D. mccartyi strains. Our results reveal a very simple mode of energy conservation in anaerobic bacteria, showing that proton translocation coupled to periplasmic electron flow might have importance also in other microbial processes and biotechnological applications.

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.

Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis.

Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal -1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.

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.

Enhanced perchloroethene dechlorination by humic acids via increasing the dehalogenase activity of Dehalococcoides strains.

Perchloroethene (PCE) is a widely used chlorinated solvent. PCE is toxic to humans and has been identified as an environmental contaminant at thousands of sites worldwide. Several Dehalococcoides mccartyi strains can transform PCE to ethene, and thus contribute to bioremediation of contaminated sites. Humic acids (HA) are ubiquitous redox-active compounds of natural aquatic and soil systems and have been intensively studied because of their effect in electron transfer. In this study, we observed the dechlorination of PCE was accelerated by HA in mixed cultures containing Dehalococcoides strains. Anthraquinone-2,6-disulfonic acid (AQDS), a humic acid analogue, inhibited PCE dechlorination in our cultures and thus induced an opposite effect on PCE dehalogenation than HA. We observed the same effect on PCE dechlorination with the pure culture of Dehalococcoides mccartyi strain CBDB1. Not only in mixed cultures but also in pure cultures, growth of Dehalococcoides was not influenced by HA but inhibited by AQDS. Enzymatic activity tests confirmed the dehalogenating activity of strain CBDB1 was increased by HA, especially when using hydrogen as electron donor. We conclude that HA enhanced PCE dechlorination by increasing the reaction speed between hydrogen and the dehalogenase enzyme rather than acting as electron shuttle through its quinone moieties.

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.