scholarly journals Structural and Functional Characterization of an Electron Transfer Flavoprotein Involved in Toluene Degradation in Strictly Anaerobic Bacteria

2019 ◽  
Vol 201 (21) ◽  
Author(s):  
Marian Samuel Vogt ◽  
Karola Schühle ◽  
Sebastian Kölzer ◽  
Patrick Peschke ◽  
Nilanjan Pal Chowdhury ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Conformational Changes ◽  
Functional Characterization ◽  
Anaerobic Bacteria ◽  
Biochemical Properties ◽  
Strictly Anaerobic ◽  
Toluene Degradation ◽  
Geobacter Metallireducens ◽  
Content Type ◽  
Facultatively Anaerobic

ABSTRACT (R)-Benzylsuccinate is the characteristic initial intermediate of anaerobic toluene metabolism, which is formed by a radical-type addition of toluene to fumarate. Its further degradation proceeds by activation to the coenzyme A (CoA)-thioester and β-oxidation involving a specific (R)-2-benzylsuccinyl-CoA dehydrogenase (BbsG) affiliated with the family of acyl-CoA dehydrogenases. In this report, we present the biochemical properties of electron transfer flavoproteins (ETFs) from the strictly anaerobic toluene-degrading species Geobacter metallireducens and Desulfobacula toluolica and the facultatively anaerobic bacterium Aromatoleum aromaticum. We determined the X-ray structure of the ETF paralogue involved in toluene metabolism of G. metallireducens, revealing strong overall similarities to previously characterized ETF variants but significantly different structural properties in the hinge regions mediating conformational changes. We also show that all strictly anaerobic toluene degraders utilize one of multiple genome-encoded related ETF paralogues, which constitute a distinct clade of similar sequences in the ETF family, for β-oxidation of benzylsuccinate. In contrast, facultatively anaerobic toluene degraders contain only one ETF species, which is utilized in all β-oxidation pathways. Our phylogenetic analysis of the known sequences of the ETF family suggests that at least 36 different clades can be differentiated, which are defined either by the taxonomic group of the respective host species (e.g., clade P for Proteobacteria) or by functional specialization (e.g., clade T for anaerobic toluene degradation). IMPORTANCE This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological electron acceptor for benzylsuccinyl-CoA dehydrogenase. While toluene-degrading denitrifying proteobacteria use a common ETF species, which is also used for other β-oxidation pathways, obligately anaerobic sulfate- or ferric-iron-reducing bacteria use specialized ETF paralogues for toluene degradation. Based on the structure and sequence conservation of these ETFs, they form a new clade that is only remotely related to the previously characterized members of the ETF family. An exhaustive analysis of the available sequences indicated that the protein family consists of several closely related clades of proven or potential electron-bifurcating ETF species and many deeply branching nonbifurcating clades, which either follow the host phylogeny or are affiliated according to functional criteria.

Application of commercial test-systems to identify gram-negative facultatively anaerobic bacteria

2016 ◽  
Vol 3 (1) ◽  
pp. 43-48 ◽  
Author(s):  
V. Patyka ◽  
L. Butsenko ◽  
L. Pasichnyk
Keyword(s):  
Erwinia Amylovora ◽  
Anaerobic Bacteria ◽  
Biochemical Properties ◽  
Test System ◽  
Phytopathogenic Bacteria ◽  
Gram Negative ◽  
Test Systems ◽  
Facultatively Anaerobic ◽  
Microbiological Methods

Aim. To validate the suitability of commercial API 20E test-system (bioMerieux) for the identifi cation and characterization of facultative gram-negative phytopathogenic bacterial isolates. Methods. Conventional mi- crobiological methods, API 20E test-system (bioMerieux) according to the manufacturer’s instructions. Re- sults. The identifi cation results for Erwinia amylovora, Pectobacterium carotovorum and Pantoea agglome- rans isolates were derived from the conventional and API 20E test systems, which, were in line with the literature data for these species. The API 20E test-system showed high suitability for P. agglomerans isolates identifi cation. Although not all the species of facultatively anaerobic phytopathogenic bacteria may be identi- fi ed using API 20E test-system, its application will surely allow obtaining reliable data about their physiologi- cal and biochemical properties, valuable for identifi cation of bacteria, in the course of 24 h. Conclusions. The results of tests, obtained for investigated species while using API 20E test-system, and those of conventional microbiological methods coincided. The application of API 20E test-system (bioMerieux) ensures fast obtain- ing of important data, which may be used to identify phytopathogenic bacteria of Erwinia, Pectobacterium, Pantoea genera.

scholarly journals Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri

2014 ◽  
Vol 80 (15) ◽  
pp. 4599-4605 ◽  
Author(s):  
Amelia-Elena Rotaru ◽  
Pravin Malla Shrestha ◽  
Fanghua Liu ◽  
Beatrice Markovaite ◽  
Shanshan Chen ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Model Organism ◽  
Methanosarcina Barkeri ◽  
Physical Contact ◽  
Acetate Metabolism ◽  
Geobacter Metallireducens ◽  
Content Type ◽  
Direct Interspecies Electron Transfer ◽  
Interspecies Electron Transfer ◽  
Effective Form

ABSTRACTDirect interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability ofMethanosarcina barkerito participate in DIET was evaluated in coculture withGeobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficientG. metallireducensstrain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficientG. metallireducensisolates to share electrons withM. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. WhenM. barkeriwas grown in coculture with the H2-producingPelobacter carbinolicus, incapable of DIET,M. barkeriutilized H2as an electron donor but metabolized little of the acetate thatP. carbinolicusproduced. This suggested that H2, but not electrons derived from DIET, inhibited acetate metabolism.P. carbinolicus-M. barkericocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H2transfer.M. barkeriis the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H2or electrons derived from DIET for CO2reduction. Furthermore,M. barkeriis genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

scholarly journals How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Nicolas Kint ◽  
Carolina Alves Feliciano ◽  
Maria C. Martins ◽  
Claire Morvan ◽  
Susana F. Fernandes ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Anaerobic Bacteria ◽  
Growth Defect ◽  
Strictly Anaerobic ◽  
Low Oxygen ◽  
Air Exposure ◽  
Vegetative Cells ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

scholarly journals Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria

2009 ◽  
Vol 191 (13) ◽  
pp. 4401-4409 ◽  
Author(s):  
Simon Wischgoll ◽  
Martin Taubert ◽  
Franziska Peters ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Oxidative Decarboxylation ◽  
Kinetic Properties ◽  
Strictly Anaerobic ◽  
Amino Acid Residues ◽  
Geobacter Metallireducens ◽  
Sulfate Reducing ◽  
Reverse Transcription Pcr ◽  
Gene Coding

ABSTRACT In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO2. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms Geobacter metallireducens (GDHGeo) (Fe[III] reducing) and Desulfococcus multivorans (GDHDes) (sulfate reducing). GDHGeo was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDHDes was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDHGeo catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDHDes catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDHGeo, the Km was 30 ± 2 μM and the V max was 3.2 ± 0.2 μmol min−1 mg−1, and for GDHDes, the Km was 52 ± 5 μM and the V max was 11 ± 1 μmol min−1 mg−1. GDHDes but not GDHGeo was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDHGeo but are missing in GDHDes. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.

scholarly journals Cyclohexa-1,5-Diene-1-Carbonyl-Coenzyme A (CoA) Hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a Common Benzoyl-CoA Degradation Pathway in Facultative and Strict Anaerobes

2006 ◽  
Vol 189 (3) ◽  
pp. 1055-1060 ◽  
Author(s):  
Franziska Peters ◽  
Yoshifumi Shinoda ◽  
Michael J. McInerney ◽  
Matthias Boll
Keyword(s):  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Strictly Anaerobic ◽  
Significant Activity ◽  
Geobacter Metallireducens ◽  
Sulfate Reducing ◽  
Coa Reductase ◽  
Syntrophus Aciditrophicus

ABSTRACT In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of β-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km , 80 and 35 μM; V max, 350 and 550 μmol min−1 mg−1, respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

scholarly journals An Aerobic Hybrid Phthalate Degradation Pathway via Phthaloyl-Coenzyme A in Denitrifying Bacteria

2020 ◽  
Vol 86 (11) ◽  
Author(s):  
Christa Ebenau-Jehle ◽  
Christina I. S. L. Soon ◽  
Jonathan Fuchs ◽  
Robin Geiger ◽  
Matthias Boll
Keyword(s):  
Phthalic Acid ◽  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Degradation Pathway ◽  
Phthalic Acid Esters ◽  
Content Type ◽  
Facultatively Anaerobic ◽  
Oxygen Sensitive ◽  
Acid Esters ◽  
Facultatively Anaerobic Bacteria

ABSTRACT The degradation of the xenobiotic phthalic acid esters by microorganisms is initiated by the hydrolysis to the respective alcohols and ortho-phthalate (hereafter, phthalate). In aerobic bacteria and fungi, oxygenases are involved in the conversion of phthalate to protocatechuate, the substrate for ring-cleaving dioxygenases. In contrast, anaerobic bacteria activate phthalate to the extremely unstable phthaloyl-coenzyme A (CoA), which is decarboxylated by oxygen-sensitive UbiD-like phthaloyl-CoA decarboxylase (PCD) to the central benzoyl-CoA intermediate. Here, we demonstrate that the facultatively anaerobic, denitrifying Thauera chlorobenzoica 3CB-1 and Aromatoleum evansii KB740 strains use phthalate as a growth substrate under aerobic and denitrifying conditions. In vitro assays with extracts from cells grown aerobically with phthalate demonstrated the succinyl-CoA-dependent activation of phthalate followed by decarboxylation to benzoyl-CoA. In T. chlorobenzoica 3CB-1, we identified PCD as a highly abundant enzyme in both aerobically and anaerobically grown cells, whereas genes for phthalate dioxygenases are missing in the genome. PCD was highly enriched from aerobically grown T. chlorobenzoica cells and was identified as an identical enzyme produced under denitrifying conditions. These results indicate that the initial steps of aerobic phthalate degradation in denitrifying bacteria are accomplished by the anaerobic enzyme inventory, whereas the benzoyl-CoA oxygenase-dependent pathway is used for further conversion to central intermediates. Such a hybrid pathway requires intracellular oxygen homeostasis at concentrations low enough to prevent PCD inactivation but sufficiently high to supply benzoyl-CoA oxygenase with its cosubstrate. IMPORTANCE Phthalic acid esters (PAEs) are industrially produced on a million-ton scale per year and are predominantly used as plasticizers. They are classified as environmentally relevant xenobiotics with a number of adverse health effects, including endocrine-disrupting activity. Biodegradation by microorganisms is considered the most effective process to eliminate PAEs from the environment. It is usually initiated by the hydrolysis of PAEs to alcohols and o-phthalic acid. Degradation of o-phthalic acid fundamentally differs in aerobic and anaerobic microorganisms; aerobic phthalate degradation heavily depends on dioxygenase-dependent reactions, whereas anaerobic degradation employs the oxygen-sensitive key enzyme phthaloyl-CoA decarboxylase. We demonstrate that aerobic phthalate degradation in facultatively anaerobic bacteria proceeds via a previously unknown hybrid degradation pathway involving oxygen-sensitive and oxygen-dependent key enzymes. Such a strategy is essential for facultatively anaerobic bacteria that frequently switch between oxic and anoxic environments.

scholarly journals Amphibacillus jilinensis sp. nov., a facultatively anaerobic, alkaliphilic bacillus from a soda lake

2010 ◽  
Vol 60 (11) ◽  
pp. 2540-2543 ◽  
Author(s):  
Xiao-Yue Wu ◽  
Gang Zheng ◽  
Wen-Wu Zhang ◽  
Xue-Wei Xu ◽  
Min Wu ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Soda Lake ◽  
Soluble Starch ◽  
Biochemical Properties ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Positive Staining ◽  
Strictly Anaerobic ◽  
Dna Relatedness ◽  
Facultatively Anaerobic

A facultatively anaerobic, alkaliphilic, spore-forming, Gram-positive-staining rod, designated Y1T, was isolated under strictly anaerobic conditions from sediment of a soda lake in Jilin province, China. The strain was not dependent on Na+ but was highly halotolerant and grew optimally in medium JY with 0.5 M Na+ (0.06 M NaHCO3 and 0.44 M NaCl). The optimum pH for growth was 9.0, with a range of pH 7.5–10.5. No growth occurred at pH 7.0 or 11.0. The strain was mesophilic, with a temperature range of 15–45 °C and optimum growth at 32 °C. Strain Y1T was able to use certain mono- and oligosaccharides. Soluble starch and casein were hydrolysed. The methyl red test, Voges–Proskauer test and tests for catalase and oxidase activities were negative. The predominant fatty acids were anteiso-C15 : 0 and iso-C15 : 0. Comparative 16S rRNA gene sequence analysis revealed 93.4–96.8 % sequence similarity to members of the genus Amphibacillus. The DNA G+C content was 37.7 mol% (T m method). The DNA–DNA relatedness of strain Y1T with respect to Amphibacillus tropicus DSM 13870T and Amphibacillus sediminis DSM 21624T was 48 and 37 %, respectively. On the basis of its phylogenetic position and the DNA–DNA relatedness data as well as its physiological and biochemical properties, strain Y1T represents a novel species of the genus Amphibacillus, for which the name Amphibacillus jilinensis sp. nov. is proposed. The type strain is Y1T (=CGMCC 1.5123T =JCM 16149T).

scholarly journals Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model

2015 ◽  
Vol 82 (4) ◽  
pp. 1080-1089 ◽  
Author(s):  
Dorothee Tegtmeier ◽  
Claire L. Thompson ◽  
Christine Schauer ◽  
Andreas Brune
Keyword(s):  
Bacterial Colonization ◽  
Anaerobic Bacteria ◽  
Close Relative ◽  
Bacterial Strains ◽  
Fermentation Products ◽  
Content Type ◽  
Facultatively Anaerobic ◽  
Pure Cultures ◽  
Metabolic Activities

ABSTRACTThe gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroachShelfordella lateralisas a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches. Fluorescence microscopy revealed that both strains specifically colonized the germfree hindgut. In diassociated cockroaches, the facultatively anaerobic strain EbSL (a new species ofEnterobacteriaceae) always outnumbered the obligately anaerobic strain FuSL (a close relative ofFusobacterium varium), irrespective of the sequence of inoculation, which showed that precolonization by facultatively anaerobic bacteria does not necessarily favor colonization by obligate anaerobes. Comparison of the fermentation products of the cultures formedin vitrowith those accumulatedin situindicated that the gut environment strongly affected the metabolic activities of both strains. The pure cultures formed the typical products of mixed-acid or butyrate fermentation, whereas the guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Similar shifts toward more-oxidized products were observed when the pure cultures were exposed to oxygen, which corroborated the strong effects of oxygen on the metabolic fluxes previously observed in termite guts. Oxygen microsensor profiles of the guts of germfree, gnotobiotic, and conventional cockroaches indicated that both gut tissue and microbiota contribute to oxygen consumption and suggest that the oxygen status influences the colonization success.

scholarly journals Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms

2009 ◽  
Vol 75 (11) ◽  
pp. 3484-3491 ◽  
Author(s):  
Michaela Stieglmeier ◽  
Reinhard Wirth ◽  
Gerhard Kminek ◽  
Christine Moissl-Eichinger
Keyword(s):  
Anaerobic Bacteria ◽  
Strictly Anaerobic ◽  
Planetary Protection ◽  
Facultatively Anaerobic ◽  
Rich Media ◽  
Biodiversity Study ◽  
Assembly Operations ◽  
First Time ◽  
Facultatively Anaerobic Bacteria ◽  
Autotrophic Microorganisms

ABSTRACT In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (Clostridium and Propionibacterium) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus.

scholarly journals Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds

2018 ◽  
Vol 200 (10) ◽  
Author(s):  
Saroj Poudel ◽  
Daniel R. Colman ◽  
Kathryn R. Fixen ◽  
Rhesa N. Ledbetter ◽  
Yanning Zheng ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Nitrogen Fixation ◽  
Energy Metabolism ◽  
Host Cells ◽  
Content Type ◽  
Anoxygenic Phototrophs ◽  
Facultatively Anaerobic ◽  
Facultative Anaerobes ◽  
Ion Translocation ◽  
Key Enzyme

ABSTRACTNitrogenase 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 N2fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O2or 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 N2reduction. 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.IMPORTANCENitrogen 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 O2or 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 theRhodobacternitrogen fixation (Rnf) complex.

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