Complete genome sequence of a haloalkaliphilic heterotrophic bacterium Halomonas salifodinae IM328.

The genome of a halophilic bacterium Halomonas salifodinae IM328 was completely sequenced in order to offer convenience for the research such as the synthesis of compatible solutes. The genome contains a circular chromosome which was sequenced by PacBio system.

Rational engineering of Halomonas salifodinae to enhance hydroxyectoine production under lower-salt conditions.

Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: • Hydroxyectoine production in H. salifodinae correlates with the salinity of medium • Transcriptomic analysis reveals the limiting factors for hydroxyectoine production • The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.

Electrochemically coupled CH4 and CO2 consumption driven by microbial processes.

The chemical transformations of methane (CH4) and carbon dioxide (CO2) greenhouse gases typically have high energy barriers. Here we present an approach of strategic coupling of CH4 oxidation and CO2 reduction in a switched microbial process governed by redox cycling of iron minerals under temperate conditions. The presence of iron minerals leads to an obvious enhancement of carbon fixation, with the minerals acting as the electron acceptor for CH4 oxidation and the electron donor for CO2 reduction, facilitated by changes in the mineral structure. The electron flow between the two functionally active microbial consortia is tracked through electrochemistry, and the energy metabolism in these consortia is predicted at the genetic level. This study offers a promising strategy for the removal of CH4 and CO2 in the natural environment and proposes an engineering technique for the utilization of major greenhouse gases.

Rice N-biofertilization by inoculation with an engineered photosynthetic diazotroph.

Photosynthetic diazotrophs expressing iron-only (Fe-only) nitrogenase can be developed into a promising biofertilizer, as it is independent on the molybdenum availability in the soil. However, the expression of Fe-only nitrogenase in diazotrophs is repressed by the fixed nitrogen of the soil, limiting the efficiency of nitrogen fixation in farmland with low ammonium concentrations that are inadequate for sustainable crop growth. Here, we succeeded in constitutively expressing the Fe-only nitrogenase even in the presence of ammonium by controlling the transcription of Fe-only nitrogenase gene cluster (anfHDGK) with the transcriptional activator of Mo nitrogenase (NifA*) in several different ways, indicating that the engineered NifA* strains can be used as promising chassis cells for efficient expression of different types of nitrogenases. When applied as a biofertilizer, the engineered Rhodopseudomonas palustris effectively stimulated rice growth, contributing to the reduced use of chemical fertilizer and the development of sustainable agriculture.

Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights.

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

EGCG-NPs inhibition HO-1-mediated reprogram iron metabolism against ferroptosis after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH), a devastating disease with a high mortality rate and poor outcomes, tightly associated with the dysregulation of iron metabolism and ferroptosis. (-)-Epigallocatechin-3-gallate (EGCG) is one of major bioactive compounds of tea catechin because of its well-known iron-chelating and antioxidative activities. However, the findings of iron-induced cell injuries after SAH remain controversial and the underlying therapeutic mechanisms of EGCG in ferroptosis is limited. Here, the ability of EGCG to inhibit iron-induced cell death following the alleviation of neurological function deficits was investigated by using in vivo SAH models. As expected, EGCG inhibited oxyhemoglobin (OxyHb)-induced the over-expression of HO-1, which mainly distributed in astrocytes and microglial cells. Subsequently, EGCG blocked ferrous iron accumulation through HO-1-mediated iron metabolic reprogramming. Therefore, oxidative stress and mitochondrial dysfunction was rescued by EGCG, which resulted in the downregulation of ferroptosis and ferritinophagy rather than apoptosis after SAH. As a result, EGCG exerted the superior therapeutic effects in the maintenance of iron homeostasis in glial cells, such as astrocytes and microglial cells, as well as in the improvement of functional outcomes after SAH. These findings highlighted that glial cells were not only the iron-rich cells in the brain but also susceptible to ferroptosis and ferritinophagy after SAH. The detrimental role of HO-1-mediated ferroptosis in glial cells can be regarded as an effective therapeutic target of EGCG in the prevention and treatment of SAH.

The Role of Glutamine Synthetase in Regulating Ammonium Assimilation and Iron-Only Nitrogenase Expression in a Photosynthetic Diazotroph.

Glutamine synthetase (GS) is responsible for the ammonium assimilation into glutamine, which serves as an important nitrogen donor for the synthesis of biomolecules and also plays a key role in regulating the nitrogen fixation catalyzed by nitrogenase. Rhodopseudomonas palustris, whose genome encodes 4 putative GSs and 3 nitrogenases, is an attractive photosynthetic diazotroph for studies of nitrogenase regulation, as it can produce the powerful greenhouse gas (methane) by iron-only (Fe-only) nitrogenase using light energy. However, the primary GS enzyme for ammonium assimilation and its role in nitrogenase regulation remain elusive in R. palustris. Here, we show that GlnA1, whose activity is finely regulated by reversible adenylylation/deadenylylation of Tyr398 residue, is primarily responsible for ammonium assimilation as the preferred GS in R. palustris. The inactivation of GlnA1 makes R. palustris shift to use the alternative GlnA2 for ammonium assimilation, resulting in the expression of Fe-only nitrogenase even in the presence of ammonium. We present a model, showing how R. palustris responds to ammonium availability and further regulates the expression of Fe-only nitrogenase. These data may contribute to the design of promising strategies for a better control of greenhouse gas emissions. IMPORTANCE The photosynthetic diazotrophs, such as Rhodopseudomonas palustris, can utilize light energy to drive the conversion of carbon dioxide (CO2) to a much more powerful greenhouse gas methane (CH4) by Fe-only nitrogenase, which is strictly regulated in response to the ammonium, a substrate of glutamine synthetase for the biosynthesis of glutamine. However, the primary glutamine synthetase for ammonium assimilation and its role in nitrogenase regulation remain unclear in R. palustris. This study shows that GlnA1 is the primary glutamine synthetase for ammonium assimilation, and also plays a key role in Fe-only nitrogenase regulation in R. palustris. For the first time, a R. palustris mutant capable of expressing Fe-only nitrogenase even in the presence of ammonium is obtained by inactivation of GlnA1. A better understanding of the Fe-only nitrogenase regulation achieved in this study provide us with new insights into the efficient control of CH4 emissions.

Bioconversion of vitamin D3 to bioactive calcifediol and calcitriol as high-value compounds.

Biological catalysis is an important approach for the production of high-value-added compounds, especially for products with complex structures. Limited by the complex steps of chemical synthesis and low yields, the bioconversion of vitamin D3 (VD3) to calcifediol and calcitriol, which are natural steroid products with high added value and significantly higher biological activity compared to VD3, is probably the most promising strategy for calcifediol and calcitriol production, and can be used as an alternative method for chemical synthesis. The conversion efficiency of VD3 to calcifediol and calcitriol has continued to rise in the past few decades with the help of several different VD3 hydroxylases, mostly cytochrome P450s (CYPs), and newly isolated strains. The production of calcifediol and calcitriol can be systematically increased in different ways. Specific CYPs and steroid C25 dehydrogenase (S25DH), as VD3 hydroxylases, are capable of converting VD3 to calcifediol and calcitriol. Some isolated actinomycetes have also been exploited for fermentative production of calcifediol and calcitriol, although the VD3 hydroxylases of these strains have not been elucidated. With the rapid development of synthetic biology and enzyme engineering, quite a lot of advances in bioproduction of calcifediol and calcitriol has been achieved in recent years. Therefore, here we review the successful strategies of promoting VD3 hydroxylation and provide some perspective on how to further improve the bioconversion of VD3 to calcifediol and calcitriol.

Identification of the Biosynthetic Pathway of Glycine Betaine That Is Responsible for Salinity Tolerance in Halophilic Thioalkalivibrio versutus D301.

Thioalkalivibrio versutus D301 has been widely used in the biodesulfurization process, as it is capable of oxidizing hydrogen sulfide to elemental sulfur under strongly halo-alkaline conditions. Glycine betaine contributes to the increased tolerance to extreme environments in some of Thioalkalivibrio species. However, the biosynthetic pathway of glycine betaine in Thioalkalivibrio remained unknown. Here, we found that genes associated with nitrogen metabolism of T. versutus D301 were significantly upregulated under high-salt conditions, causing the enhanced production of glycine betaine that functions as a main compatible solute in response to the salinity stress. Glycine betaine was synthesized by glycine methylation pathway in T. versutus D301, with glycine N-methyltransferase (GMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) as key enzymes in this pathway. Moreover, substrate specificities of GMT and SDMT were quite different from the well characterized enzymes for glycine methylation in halophilic Halorhodospira halochloris. Our results illustrate the glycine betaine biosynthetic pathway in the genus of Thioalkalivibrio for the first time, providing us with a better understanding of the biosynthesis of glycine betaine in haloalkaliphilic Thioalkalivibrio.

A Red Fluorescent Protein Reporter System Developed for Measuring Gene Expression in Photosynthetic Bacteria under Anaerobic Conditions.

The photosynthetic bacterium Rhodopseudomonas palustris converts nitrogen gas (N2) to fertilizer ammonia (NH3) and also produces clean energy hydrogen gas (H2) from protons (H+) when it is grown anaerobically in nitrogen fixing medium with illumination, a condition that promotes the expression of active nitrogenase. Compared with quantitative real-time PCR (qRT-PCR) and the lacZ reporter system, two methods commonly used for in vivo study of nitrogenase regulation in photosynthetic bacteria, the fluorescent protein reporter system has advantages in terms of its simplicity and sensitivity. However, little is known concerning if the fluorescent protein reporter system can be used in bacterial cells that need to grow anaerobically. Here, we developed an RFP-based method to measure the nitrogenase gene expression in photosynthetic bacteria grown anaerobically. This method was able to determine the levels of both the genome-based and the plasmid-based nitrogenase expression under anaerobic conditions, providing a better method for in vivo study of gene expression affected by oxygen. The RFP reporter system developed here will promote a better understanding of the molecular mechanism of nitrogenase regulation and will be used on other genes of interest in a wider range of anaerobic bacteria.

[Enhanced heterologous expression of the cytochrome c from uncultured anaerobic methanotrophic archaea].

Cytochrome c is a type of heme proteins that are widely distributed in living organisms. It consists of heme and apocytochrome c, and has potential applications in bioelectronics, biomedicine and pollutant degradation. However, heterologous overexpression of cytochrome c is still challenging. To date, expression of the cytochrome c from uncultured anaerobic methanotrophic archaea has not been reported, and nothing is known about the function of this cytochrome c. A his tagged cytochrome c was successfully expressed in E. coli by introducing a thrombin at the N-terminus of CytC4 and co-expressing CcmABCDEFGH, which is responsible for the maturation of cytochrome c. Shewanella oneidensis, which naturally has enzymes for cytochrome c maturation, was then used as a host to further increase the expression of CytC4. Indeed, a significantly higher expression of CytC4 was achieved in S. oneidensis when compared with in E. coli. The successful heterologous overexpression of CytC4 will facilitate the exploitation of its physiological functions and biotechnological applications.

Development of whole-cell catalyst system for sulfide biotreatment based on the engineered haloalkaliphilic bacterium.

Microorganisms play an essential role in sulfide removal. Alkaline absorption solution facilitates the sulfide's dissolution and oxidative degradation, so haloalkaliphile is a prospective source for environmental-friendly and cost-effective biodesulfurization. In this research, 484 sulfide oxidation genes were identified from the metagenomes of the soda-saline lakes and a haloalkaliphilic heterotrophic bacterium Halomonas salifodinae IM328 (=CGMCC 22183) was isolated from the same habitat as the host for expression of a representative sequence. The genetic manipulation was successfully achieved through the conjugation transformation method, and sulfide: quinone oxidoreductase gene (sqr) was expressed via pBBR1MCS derivative plasmid. Furthermore, a whole-cell catalyst system was developed by using the engineered strain that exhibited a higher rate of sulfide oxidation under the optimal alkaline pH of 9.0. The whole-cell catalyst could be recycled six times to maintain the sulfide oxidation rates from 41.451 to 80.216 µmol·min-1·g-1 dry cell mass. To summarize, a whole-cell catalyst system based on the engineered haloalkaliphilic bacterium is potentiated to be applied in the sulfide treatment at a reduced cost.

Recent advances in the biosynthesis of isoprenoids in engineered Saccharomyces cerevisiae.

Isoprenoids, as the largest group of chemicals in the domains of life, constitute more than 50,000 members. These compounds consist of different numbers of isoprene units (C5H8), by which they are typically classified into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30), and tetraterpenoids (C40). In recent years, isoprenoids have been employed as food additives, in the pharmaceutical industry, as advanced biofuels, and so on. To realize the sufficient and efficient production of valuable isoprenoids on an industrial scale, fermentation using engineered microorganisms is a promising strategy compared to traditional plant extraction and chemical synthesis. Due to the advantages of mature genetic manipulation, robustness and applicability to industrial bioprocesses, Saccharomyces cerevisiae has become an attractive microbial host for biochemical production, including that of various isoprenoids. In this review, we summarized the advances in the biosynthesis of isoprenoids in engineered S. cerevisiae over several decades, including synthetic pathway engineering, microbial host engineering, and central carbon pathway engineering. Furthermore, the challenges and corresponding strategies towards improving isoprenoid production in engineered S. cerevisiae were also summarized. Finally, suggestions and directions for isoprenoid production in engineered S. cerevisiae in the future are discussed.

Microbial production of ectoine and hydroxyectoine as high-value chemicals.

Ectoine and hydroxyectoine as typical representatives of compatible solutes are not only essential for extremophiles to survive in extreme environments, but also widely used in cosmetic and medical industries. Ectoine was traditionally produced by Halomonas elongata through a "bacterial milking" process, of which the marked feature is using a high-salt medium to stimulate ectoine biosynthesis and then excreting ectoine into a low-salt medium by osmotic shock. The optimal hydroxyectoine production was achieved by optimizing the fermentation process of Halomonas salina. However, high-salinity broth exacerbates the corrosion to fermenters, and more importantly, brings a big challenge to the subsequent wastewater treatment. Therefore, increasing attention has been paid to reducing the salinity of the fermentation broth but without a sacrifice of ectoine/hydroxyectoine production. With the fast development of functional genomics and synthetic biology, quite a lot of progress on the bioproduction of ectoine/hydroxyectoine has been achieved in recent years. The importation and expression of an ectoine producing pathway in a non-halophilic chassis has so far achieved the highest titer of ectoine (~ 65 g/L), while rational flux-tuning of halophilic chassis represents a promising strategy for the next-generation of ectoine industrial production. However, efficient conversion of ectoine to hydroxyectoine, which could benefit from a clearer understanding of the ectoine hydroxylase, is still a challenge to date.

Abundant Taxa and Favorable Pathways in the Microbiome of Soda-Saline Lakes in Inner Mongolia.

Soda-saline lakes are a special type of alkaline lake in which the chloride concentration is greater than the carbonate/bicarbonate concentration. Due to the high pH and a usually higher osmotic pressure than that of a normal soda lake, the microbes may need more energy to thrive in such a double-extreme environment. In this study, we systematically investigated the microbiome of the brine and sediment samples of nine artificially separated ponds (salinities from 5.5% to saturation) within two soda-saline lakes in Inner Mongolia of China, assisted by deep metagenomic sequencing. The main inorganic ions shaped the microbial community in both the brines and sediments, and the chloride concentration exhibited the most significant effect. A total of 385 metagenome-assembled genomes (MAGs) were generated, in which 38 MAGs were revealed as the abundant species in at least one of the eighteen different samples. Interestingly, these abundant species also represented the most branches of the microbiome of the soda-saline lakes at the phylum level. These abundant taxa were close relatives of microorganisms from classic soda lakes and neutral saline environments, but forming a combination of both habitats. Notably, approximately half of the abundant MAGs had the potential to drive dissimilatory sulfur cycling. These MAGs included four autotrophic Ectothiorhodospiraceae MAGs, one Cyanobacteria MAG and nine heterotrophic MAGs with the potential to oxidize sulfur, as well as four abundant MAGs containing genes for elemental sulfur respiration. The possible reason is that reductive sulfur compounds could provide additional energy for the related species, and reductions of oxidative sulfur compounds are more prone to occur under alkaline conditions which support the sulfur cycling. In addition, a unique 1,4-alpha-glucan phosphorylation pathway, but not a normal hydrolysis one, was found in the abundant Candidatus Nanohaloarchaeota MAG NHA-1, which would produce more energy in polysaccharide degradation. In summary, this work has revealed the abundant taxa and favorable pathways in the soda-saline lakes, indicating that efficient energy regeneration pathway may increase the capacity for environmental adaptation in such saline-alkaline environments. These findings may help to elucidate the relationship between microbial metabolism and adaptation to extreme environments.

Facile Preparation of Stable Solid-State Carbon Quantum Dots with Multi-Peak Emission.

Aggregation-caused quenching (ACQ) effect, known as the main cause to restrain solid-state luminescence of carbon quantum dots (CQDs), hinders further application of CQDs in white light-emitting diodes (WLED). Here, a complex of CQDs and phthalimide crystals (CQDs/PC) was prepared through a one-step solvothermal method. CQDs/PC prevented CQDs from touching directly by embedding the CQDs in phthalimide crystal matrix in situ, which effectively reduced the ACQ effect. Furthermore, CQDs/PC exhibited multi-peak fluorescence spectra that span the green, yellow and orange spectral regions. Finally, a WLED fabricated based on CQDs/PC achieved a color-rendering index of 82 and a correlated color temperature of 5430 K. This work provides a quick and effective strategy to apply CQDs to WLED.

Effective Efficiency Advantage Assessment of Information Filter for Conventional Kalman Filter in GNSS Scenarios.

The Global Navigation Satellite System (GNSS) is a widely used positioning technique. Computational efficiency is crucial to applications such as real-time GNSS positioning and GNSS network data processing. Many researchers have made great efforts to address this problem by means such as parameter elimination or satellite selection. However, parameter estimation is rarely discussed when analyzing GNSS algorithm efficiency. In addition, most studies on Kalman filter (KF) efficiency commonly have defects, such as neglecting application-specified optimization and limiting specific hardware platforms in the conclusion. The former reduces the practicality of the solution, because applications that need such analyses on filters are often optimized, and the latter reduces its generality because of differences between platforms. In this paper, the computational cost enhancement of replacing the conventional KF with the information filter (IF) is tested considering GNSS application-oriented optimization conditions and hardware platform differences. First, optimization conditions are abstracted from GNSS data-processing scenarios. Then, a thorough analysis is carried out on the computational cost of the filters, considering hardware-platform differences. Finally, a case of GNSS dynamic differencing positioning is studied. The simulation shows that the IF is slightly faster for precise point positioning and much faster for the code-based single-difference GNSS (SDGNSS) with the constant velocity (CV) model than the conventional KF, but is not a good substitute for the conventional KF in the other algorithms mentioned. The real test shows that the IF is about 50% faster than the conventional KF handling code-based SDGNSS with the CV model. Also, the information filter is theoretically equivalent to and can produce results that are consistent with the Kalman filter. Our conclusions can be used as a reference for GNSS applications that need high process speed or real-time capability.

Influence of Energy and Electron Availability on In Vivo Methane and Hydrogen Production by a Variant Molybdenum Nitrogenase.

The anoxygenic phototrophic bacterium Rhodopseudomonas palustris produces methane (CH4) from carbon dioxide (CO2) and hydrogen (H2) from protons (H+) when it expresses a variant form of molybdenum (Mo) nitrogenase that has two amino acid substitutions near its active site. We examined the influence of light energy and electron availability on in vivo production of these biofuels. Nitrogenase activity requires large amounts of ATP, and cells exposed to increasing light intensities produced increasing amounts of CH4 and H2 As expected for a phototroph, intracellular ATP increased with increasing light intensity, but there was only a loose correlation between ATP content and CH4 and H2 production. There was a much stronger correlation between decreased intracellular ADP and increased gas production with increased light intensity, suggesting that the rate-limiting step for CH4 and H2 production by R. palustris is inhibition of nitrogenase by ADP. Increasing the amounts of electrons available to nitrogenase by providing cells with organic alcohols, using nongrowing cells, blocking electrons from entering the Calvin cycle, or blocking H2 uptake resulted in higher yields of H2 and, in some cases, CH4 Our results provide a more complete understanding of the constraints on nitrogenase-based production of biofuels.IMPORTANCE A variant form of Mo nitrogenase catalyzes the conversion of CO2 and protons to the biofuels CH4 and H2 A constant supply of electrons and ATP is needed to drive these reduction reactions. The bacterium R. palustris generates ATP from light and has a versatile metabolism that makes it ideal for manipulating electron availability intracellularly. We therefore explored its potential as a biocatalyst for CH4 and H2 production. We found that intracellular ADP had a major effect on biofuel production, more pronounced than the effect caused by ATP. This is probably due to inhibition of nitrogenase activity by ADP. In general, the amount of CH4 produced by the variant nitrogenase in vivo was affected by electron availability much less than was the amount of H2 produced. This study shows the nature of constraints on in vivo biofuel production by variant Mo nitrogenase.

Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds.

Nitrogenase catalyzes the reduction of dinitrogen (N2) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2)-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.

A pathway for biological methane production using bacterial iron-only nitrogenase.

Methane (CH4) is a potent greenhouse gas that is released from fossil fuels and is also produced by microbial activity, with at least one billion tonnes of CH4 being formed and consumed by microorganisms in a single year 1 . Complex methanogenesis pathways used by archaea are the main route for bioconversion of carbon dioxide (CO2) to CH4 in nature2-4. Here, we report that wild-type iron-iron (Fe-only) nitrogenase from the bacterium Rhodopseudomonas palustris reduces CO2 simultaneously with nitrogen gas (N2) and protons to yield CH4, ammonia (NH3) and hydrogen gas (H2) in a single enzymatic step. The amount of CH4 produced by purified Fe-only nitrogenase was low compared to its other products, but CH4 production by this enzyme in R. palustris was sufficient to support the growth of an obligate CH4-utilizing Methylomonas strain when the two microorganisms were grown in co-culture, with oxygen (O2) added at intervals. Other nitrogen-fixing bacteria that we tested also formed CH4 when expressing Fe-only nitrogenase, suggesting that this is a general property of this enzyme. The genomes of 9% of diverse nitrogen-fixing microorganisms from a range of environments encode Fe-only nitrogenase. Our data suggest that active Fe-only nitrogenase, present in diverse microorganisms, contributes CH4 that could shape microbial community interactions.