Uptake Hydrogenase Research Articles

Frankia [NiFe] uptake hydrogenases and genome reduction: different lineages of loss.

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup-, while only one Hup- Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a and 3b according to Søndergaard et al. (2016); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1h and group 3b are not.

Proteomic changes orchestrate metabolic acclimation of a unicellular diazotrophic cyanobacterium during light-dark cycle and nitrogen fixation states.

Cyanobacteria have developed an impressive array of proteins and pathways, each tailored for specific metabolic attributes, to execute photosynthesis and biological nitrogen (N2)-fixation. An understanding of these biologically incompatible processes provides important insights into how they can be optimized for renewable energy. To expand upon our current knowledge, we performed label-free quantitative proteomic analysis of the unicellular diazotrophic cyanobacterium Crocosphaera subtropica ATCC 51142 grown with and without nitrate under 12-hour light-dark cycles. Results showed significant shift in metabolic activities including photosynthesis, respiration, biological nitrogen fixation (BNF), and proteostasis to different growth conditions. We identified 14 nitrogenase enzymes which were among the most highly expressed proteins in the dark under nitrogen-fixing conditions, emphasizing their importance in BNF. Nitrogenase enzymes were not expressed under non nitrogen fixing conditions, suggesting a regulatory mechanism based on nitrogen availability. The synthesis of key respiratory enzymes and uptake hydrogenase (HupSL) synchronized with the synthesis of nitrogenase indicating a coordinated regulation of processes involved in energy production and BNF. Data suggests alternative pathways that cells utilize, such as oxidative pentose phosphate (OPP) and 2-oxoglutarate (2-OG) pathways, to produce ATP and support bioenergetic BNF. Data also indicates the important role of uptake hydrogenase for the removal of O2 to support BNF. Overall, this study expands upon our knowledge regarding molecular responses of Crocosphaera 51142 to nitrogen and light-dark phases, shedding light on potential applications and optimization for renewable energy.

Biohydrogen utilization in legume-rhizobium symbiosis reveals a novel mechanism of accelerated tetrachlorobiphenyl transformation

Symbiosis between Glycine max and Bradyrhizobium diazoefficiens were used as a model system to investigate whether biohydrogen utilization promotes the transformation of the tetrachlorobiphenyl PCB77. Both a H2 uptake-positive (Hup+) strain (wild type) and a Hup- strain (a hupL deletion mutant) were inoculated into soybean nodules. Compared with Hup- nodules, Hup+ nodules increased dechlorination significantly by 61.1 % and reduced the accumulation of PCB77 in nodules by 37.7 % (p < 0.05). After exposure to nickel, an enhancer of uptake hydrogenase, dechlorination increased significantly by 2.2-fold, and the accumulation of PCB77 in nodules decreased by 54.4 % (p < 0.05). Furthermore, the tetrachlorobiphenyl transformation in the soybean root nodules was mainly testified to be mediated by nitrate reductase (encoded by the gene NR) for tetrachlorobiphenyl dechlorination and biphenyl-2,3-diol 1,2-dioxygenase (bphC) for biphenyl degradation. This study demonstrates for the first time that biohydrogen utilization has a beneficial effect on tetrachlorobiphenyl biotransformation in a legume-rhizobium symbiosis.

Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert.

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H2 and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.IMPORTANCEThe ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment-including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

Understanding microbiome dynamics and functional responses during acidogenic fermentation of sucrose and sugarcane vinasse through metatranscriptomic analysis

Improving anaerobic digestion of sugarcane vinasse – a high-strength wastewater from ethanol distillation – is a subject of great interest, in view of the reduction of the pollutants and recovery of methane and valuable metabolites as byproducts. Through metatranscriptomic analysis, this study evaluated the active microbiome and metabolic pathways in a continuous acidogenic reactor: Stage 1S (control): 100% sucrose-based substrate (SBS); Stage 2SV (acclimation): 50% SBS and 50% vinasse; Stage 3V: 100% vinasse. Metatranscriptome obtained from each Stage was subjected to taxonomic and functional annotations. Under SBS feeding, pH dropped to pH 2.7 and biohydrogen production was observed. As vinasse was added, pH increased to 4.1–4.5, resulting in community structure and metabolite changes. In Stage 3V, biohydrogen production ceased, and propionate and acetate prevailed among the volatile fatty acids. Release of homoacetogenesis enzymes by Clostridium ljungdahlii and of uptake hydrogenase (EC 1.12.99.6) by Pectinatus frisingensis were linked to hydrogen consumption in Stages 2SV and 3V. Metabolic pathways of vinasse compounds, such as carbohydrates, malate, oxalate, glycerol, sulfate and phenol, were investigated in detail. In pyruvate metabolism, gene transcripts of oadA (oxaloacetate decarboxylase) and mdh (malate dehydrogenase), were upregulated in Stage 3V, being mostly attributed to P. frisingensis. Acetate formation from vinasse degradation was mainly attributed to Megasphaera and Clostridium, and propionate formation to P. frisingensis. Glycerol removal from vinasse exceeded 99%, and gene transcripts encoding for glpF (glycerol uptake facilitator protein), glpK (glycerol kinase) and glpABC (glycerol-3-phosphate dehydrogenase) were expressed mostly by Pectinatus and Prevotella. mRNA profiling showed that active bacteria and gene expression greatly changed when vinasse replaced sucrose, and Pectinatus was the main active bacterium degrading the searched compounds from vinasse. The identification of the main metabolic routes and the associated microorganisms achieved in this work contributes with valuable information to support further optimization of fermentation towards the desired metabolites.

The survival strategy of direct interspecies electron transfer-capable coculture under electron donor-limited environments

Direct interspecies electron transfer (DIET) has been considered as an effective mechanism for interspecies electron exchange in microbial syntrophy. Understanding DIET-capable syntrophic associations under energy-limited environments is important because these conditions more closely approximate those found in natural subsurface environments than in the batch cultures in the laboratory. This study, investigated the metabolic dynamics and electron transfer mechanisms in DIET-capable syntrophic coculture of Geobacter metallireducens and Geobacter sulfurreducens under electron donor-limited condition. The wild-coculture and the mutant-coculture with a citrate synthase-deficient G. sulfurreducens exhibited similar rates of syntrophic metabolism under ethanol-limited and ethanol-replete conditions. Transcriptomic analyses revealed that, in the mutant-coculture in which interspecies electron exchange was the sole electron source for G. sulfurreducens, the transcription of genes associated with uptake hydrogenase in G. sulfurreducens were significantly repressed and thus DIET tended to be the preferred mode of interspecies electron exchange under electron donor-limited condition. To overcome electron donor limitation, c-type cytochromes in the coculture actively moved from outer membrane to extracellular environment, potentially via increased secretion of outer-membrane vesicles. These results suggested a preferred electron transfer mechanism for DIET-capable syntrophic communities' survival in the electron donor-limited environments, providing valuable insights into the biogeochemical processes mediated by DIET in natural and engineered environments.

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.

Novel symbiovars ingae, lysilomae and lysilomaefficiens in bradyrhizobia from tree-legume nodules

Inga vera and Lysiloma tree legumes form nodules with Bradyrhizobium spp. from the japonicum group that represent novel genomospecies, for which we describe here using genome data, symbiovars lysilomae, lysilomaefficiens and ingae. Genes encoding Type three secretion system (TTSS) that could affect host specificity were found in ingae but not in lysilomae nor in lysilomaefficiens symbiovars and uptake hydrogenase hup genes (that affect nitrogen fixation) were observed in bradyrhizobia from the symbiovars ingae and lysilomaefficiens. nolA gene was found in the symbiovar lysilomaefficiens but not in strains from lysilomae. We discuss that multiple genes may dictate symbiosis specificity. Besides, toxin-antitoxin genes were found in the symbiosis islands in bradyrhizobia from symbiovars ingae and lysilomaefficiens. A limit (95%) to define symbiovars with nifH gene sequences was proposed here.

Mixotrophy broadens the ecological niche range of the iron oxidizerSideroxydanssp. CL21 isolated from an iron-rich peatland

Sideroxydans sp. CL21 is a microaerobic, acid-tolerant Fe(II)-oxidizer, isolated from the Schlöppnerbrunnen fen. Since the genome size of Sideroxydans sp. CL21 is 21% larger than that of the neutrophilic Sideroxydans lithotrophicus ES-1, we hypothesized that strain CL21 contains additional metabolic traits to thrive in the fen. The common genomic content of both strains contains homologs of the putative Fe(II) oxidation genes, mtoAB and cyc2. A large part of the accessory genome in strain CL21 contains genes linked to utilization of alternative electron donors, including NiFe uptake hydrogenases, and genes encoding lactate uptake and utilization proteins, motility and biofilm formation, transposable elements, and pH homeostasis mechanisms. Next, we incubated the strain in different combinations of electron donors and characterized the fen microbial communities. Sideroxydans spp. comprised 3.33% and 3.94% of the total relative abundance in the peatland soil and peatland water, respectively. Incubation results indicate Sideroxydans sp. CL21 uses H2 and thiosulfate, while lactate only enhances growth when combined with Fe, H2, or thiosulfate. Rates of H2 utilization were highest in combination with other substrates. Thus, Sideroxydans sp. CL21 is a mixotroph, growing best by simultaneously using substrate combinations, which helps to thrive in dynamic and complex habitats.

Genomic insights into the physiology of Quinella, an iconic uncultured rumen bacterium

Quinella is a genus of iconic rumen bacteria first reported in 1913. There are no cultures of these bacteria, and information on their physiology is scarce and contradictory. Increased abundance of Quinella was previously found in the rumens of some sheep that emit low amounts of methane (CH4) relative to their feed intake, but whether Quinella contributes to low CH4 emissions is not known. Here, we concentrate Quinella cells from sheep rumen contents, extract and sequence DNA, and reconstruct Quinella genomes that are >90% complete with as little as 0.20% contamination. Bioinformatic analyses of the encoded proteins indicate that lactate and propionate formation are major fermentation pathways. The presence of a gene encoding a potential uptake hydrogenase suggests that Quinella might be able to use free hydrogen (H2). None of the inferred metabolic pathways is predicted to produce H2, a major precursor of CH4, which is consistent with the lower CH4 emissions from those sheep with high abundances of this bacterium.

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments.

The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O2 -sensitive N2 -fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (a Mehler-like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative-specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is responsible for moderate O2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.

Genomic Diversity of Bradyrhizobium from the Tree Legumes Inga and Lysiloma (Caesalpinioideae-Mimosoid Clade)

We identified diverse bradyrhizobia having distinct ERIC-PCR genomic fingerprints from native American trees Inga vera and Lysiloma spp. In addition, two nodule isolates recovered from Lysiloma divaricatum seedlings inoculated with soil from the tropical house of a UK botanical garden were also identified as Bradyrhizobium. Genomes were obtained (with sizes around 9 Mb each) from nine Inga and Lysiloma bradyrhizobial isolates; plasmids were detected in two of the Inga strains analyzed. Average Nucleotide Identity of whole genomes revealed five novel Bradyrhizobium genomospecies from Mexican trees, while the UK isolates were identified as Bradyrhizobium cajani and Bradyrhizobium brasilense. Inga vera and Lysiloma isolates, despite their genetic distances and different hosts, shared a common set of nod genes that suggested that I. vera and Lysiloma bradyrhizobia produce fucosylated, methylated and carbamylated lipochitooligosaccharides. Uptake hydrogenase hup, hyp and secretion system genes were found in some of the isolates. Lysiloma strains were found to be ineffective on I. vera. Some of the isolates may be used as plant inoculants.

The effects of Ni availability on H2 production and N2 fixation in a model unicellular diazotroph: The expression of hydrogenase and nitrogenase

AbstractWe investigated the effects of Ni concentration on hydrogen (H2) net production and nitrogen (N2) fixation rates in a model unicellular diazotrophic cyanobacterium, Cyanothece. We quantified NiFe uptake hydrogenase of the diazotroph to examine how Ni deficiency affects the expression of hydrogenase and nitrogenase and N2 fixation rates. We found that Ni availability controls its H2 net production and the expression of HupL, the major subunit of the uptake hydrogenase. Low Ni availability induced low HupL expression levels while yielding high H2 net production, and vice versa. We also identified the threshold range of Ni concentrations that may differentiate low and high H2 net production patterns in the diazotroph. However, the substantial variations in Ni availability and H2 net production did not affect nitrogenase expression and N2 fixation rates in Cyanothece. From two independent studies in the Atlantic tropical and subtropical oceans, we found that dissolved Ni concentrations and H2 oversaturation percentage exhibited a statistically significant negative correlation, which is consistent with the findings of this culture study. Overall, these results indicate that Ni availability may affect H2 net production and concentrations in the euphotic zone of the oceanic regions. H2 concentrations may be used as a promising proxy to reflect N2 fixation activities in tropical and subtropical oceans only under Ni‐deficient conditions.

Proteomic Analysis of a Syntrophic Coculture of Syntrophobacter fumaroxidans MPOBT and Geobacter sulfurreducens PCAT.

We established a syntrophic coculture of Syntrophobacter fumaroxidans MPOBT (SF) and Geobacter sulfurreducens PCAT (GS) growing on propionate and Fe(III). Neither of the bacteria was capable of growth on propionate and Fe(III) in pure culture. Propionate degradation by SF provides acetate, hydrogen, and/or formate that can be used as electron donors by GS with Fe(III) citrate as electron acceptor. Proteomic analyses of the SF-GS coculture revealed propionate conversion via the methylmalonyl-CoA (MMC) pathway by SF. The possibility of interspecies electron transfer (IET) via direct (DIET) and/or hydrogen/formate transfer (HFIT) was investigated by comparing the differential abundance of associated proteins in SF-GS coculture against (i) SF coculture with Methanospirillum hungatei (SF-MH), which relies on HFIT, (ii) GS pure culture growing on acetate, formate, hydrogen as propionate products, and Fe(III). We noted some evidence for DIET in the SF-GS coculture, i.e., GS in the coculture showed significantly lower abundance of uptake hydrogenase (43-fold) and formate dehydrogenase (45-fold) and significantly higher abundance of proteins related to acetate metabolism (i.e., GltA; 62-fold) compared to GS pure culture. Moreover, SF in the SF-GS coculture showed significantly lower abundance of IET-related formate dehydrogenases, Fdh3 (51-fold) and Fdh5 (29-fold), and the rate of propionate conversion in SF-GS was 8-fold lower than in the SF-MH coculture. In contrast, compared to GS pure culture, we found lower abundance of pilus-associated cytochrome OmcS (2-fold) and piliA (5-fold) in the SF-GS coculture that is suggested to be necessary for DIET. Furthermore, neither visible aggregates formed in the SF-GS coculture, nor the pili-E of SF (suggested as e-pili) were detected. These findings suggest that the IET mechanism is complex in the SF-GS coculture and can be mediated by several mechanisms rather than one discrete pathway. Our study can be further useful in understanding syntrophic propionate degradation in bioelectrochemical and anaerobic digestion systems.

Co-production of hydrogen and ethyl acetate in Escherichia coli

BackgroundEthyl acetate (C4H8O2) and hydrogen (H2) are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products renders the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved, but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO2.ResultsWe engineered Escherichia coli towards improved conversion of formate into H2 and CO2 by inactivating the formate hydrogen lyase repressor (hycA), both uptake hydrogenases (hyaAB, hybBC) and/or overexpressing the hydrogen formate lyase activator (fhlA), in an acetate kinase (ackA) and lactate dehydrogenase (ldhA)-deficient background strain. Initially 10 strains, with increasing number of modifications were evaluated in anaerobic serum bottles with respect to growth. Four reference strains ΔldhAΔackA, ΔldhAΔackA p3-fhlA, ΔldhAΔackAΔhycAΔhyaABΔhybBC and ΔldhAΔackAΔhycAΔhyaABΔhybBC p3-fhlA were further equipped with a plasmid carrying the heterologous ethanol acyltransferase (Eat1) from Wickerhamomyces anomalus and analyzed with respect to their ethyl acetate and hydrogen co-production capacity. Anaerobic co-production of hydrogen and ethyl acetate via Eat1 was achieved in 1.5-L pH-controlled bioreactors. The cultivation was performed at 30 °C in modified M9 medium with glucose as the sole carbon source. Anaerobic conditions and gas stripping were established by supplying N2 gas.ConclusionsWe showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70% of the pathway maximum for ethyl acetate and hydrogen, and propose in situ product removal via gas stripping as efficient technique to isolate the products of interest.

Termite gas emissions select for hydrogenotrophic microbial communities in termite mounds

Organoheterotrophs are the dominant bacteria in most soils worldwide. While many of these bacteria can subsist on atmospheric hydrogen (H2), levels of this gas are generally insufficient to sustain hydrogenotrophic growth. In contrast, bacteria residing within soil-derived termite mounds are exposed to high fluxes of H2 due to fermentative production within termite guts. Here, we show through community, metagenomic, and biogeochemical profiling that termite emissions select for a community dominated by diverse hydrogenotrophic Actinobacteriota and Dormibacterota. Based on metagenomic short reads and derived genomes, uptake hydrogenase and chemosynthetic RuBisCO genes were significantly enriched in mounds compared to surrounding soils. In situ and ex situ measurements confirmed that high- and low-affinity H2-oxidizing bacteria were highly active in the mounds, such that they efficiently consumed all termite-derived H2 emissions and served as net sinks of atmospheric H2 Concordant findings were observed across the mounds of three different Australian termite species, with termite activity strongly predicting H2 oxidation rates (R2 = 0.82). Cell-specific power calculations confirmed the potential for hydrogenotrophic growth in the mounds with most termite activity. In contrast, while methane is produced at similar rates to H2 by termites, mounds contained few methanotrophs and were net sources of methane. Altogether, these findings provide further evidence of a highly responsive terrestrial sink for H2 but not methane and suggest H2 availability shapes composition and activity of microbial communities. They also reveal a unique arthropod-bacteria interaction dependent on H2 transfer between host-associated and free-living microbial communities.

Introduction of H2-Uptake Hydrogenase Genes Into Rhizobial Strains Improves Symbiotic Nitrogen Fixation in Vicia sativa and Lotus corniculatus Forage Legumes

Biological nitrogen fixation by the Rhizobium-legume symbiosis allows the conversion of atmospheric nitrogen into ammonia within root nodules mediated by the nitrogenase enzyme. Nitrogenase activity results in the evolution of hydrogen as a result of a side reaction intrinsic to the activity of this enzyme. Some rhizobia, and also other nitrogen fixers, induce a NiFe uptake hydrogenase (Hup) to recycle hydrogen produced by nitrogenase, thus improving the efficiency of the nitrogen fixation process. In this work we report the generation and symbiotic behavior of hydrogenase-positive Rhizobium leguminosarum and Mesorhizobium loti strains effective in vetch (Vicia sativa) and birsfoot trefoil (Lotus corniculatus) forage crops, respectively. The ability of hydrogen recycling was transferred to these strains through the incorporation of hup minitransposon TnHB100, thus leading to full recycling of hydrogen in nodules. Inoculation of Vicia and Lotus plants with these engineered strains led to significant increases in the levels of nitrogen incorporated into the host legumes. The level of improvement of symbiotic performance was dependent on the recipient strain and also on the legume host. These results indicate that hydrogen recycling has the potential to improve symbiotic nitrogen fixation in forage plants.

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases*.

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom‐economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25–70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme “ene‐reductases” to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min−1 and total turnover numbers up to 20 200 were recorded during flavin recycling.

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases.

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene-reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.

In-Situ Metatranscriptomic Analyses Reveal the Metabolic Flexibility of the Thermophilic Anoxygenic Photosynthetic Bacterium Chloroflexus aggregans in a Hot Spring Cyanobacteria-Dominated Microbial Mat.

Chloroflexus aggregans is a metabolically versatile, thermophilic, anoxygenic phototrophic member of the phylum Chloroflexota (formerly Chloroflexi), which can grow photoheterotrophically, photoautotrophically, chemoheterotrophically, and chemoautotrophically. In hot spring-associated microbial mats, C. aggregans co-exists with oxygenic cyanobacteria under dynamic micro-environmental conditions. To elucidate the predominant growth modes of C. aggregans, relative transcription levels of energy metabolism- and CO2 fixation-related genes were studied in Nakabusa Hot Springs microbial mats over a diel cycle and correlated with microscale in situ measurements of O2 and light. Metatranscriptomic analyses indicated two periods with different modes of energy metabolism of C. aggregans: (1) phototrophy around midday and (2) chemotrophy in the early morning hours. During midday, C. aggregans mainly employed photoheterotrophy when the microbial mats were hyperoxic (400–800 µmol L−1 O2). In the early morning hours, relative transcription peaks of genes encoding uptake hydrogenase, key enzymes for carbon fixation, respiratory complexes as well as enzymes for TCA cycle and acetate uptake suggest an aerobic chemomixotrophic lifestyle. This is the first in situ study of the versatile energy metabolism of C. aggregans based on gene transcription patterns. The results provide novel insights into the metabolic flexibility of these filamentous anoxygenic phototrophs that thrive under dynamic environmental conditions.