Direct cathodic electron uptake coupled to sulfate reduction by Desulfovibrio ferrophilus IS5 biofilms

Direct electron uptake is emerging as a key process for electron transfer in anaerobic microbial communities, both between species and from extracellular sources, such as zero-valent iron (Fe⁰) or cathodic surfaces. In this study, we investigated cathodic electron uptake by Fe⁰-corroding Desulfovibrio ferrophilus IS5 and showed that electron uptake is dependent on direct cell contact via a biofilm on the cathode surface rather than through secreted intermediates. Induction of cathodic electron uptake by lactate-starved D. ferrophilus IS5 cells resulted in the expression of all components necessary for electron uptake; however, protein synthesis was required for full biofilm formation. Notably, proteinase K treatment uncoupled electron uptake from biofilm formation, likely through proteolytic degradation of proteinaceous components of the electron uptake machinery. We also showed that cathodic electron uptake is dependent on SO₄²⁻ reduction. The insensitivity of Fe⁰ corrosion to proteinase K treatment suggests that electron uptake from a cathode might involve different mechanism(s) than those involved in Fe⁰ corrosion.     

Uptake hydrogenase activity in denitrifying Azospirillum brasilense grown anaerobically with nitrous oxide or nitrate.

zospirillum brasilense Sp7 was grown anaerobically with N2O as the terminal electron acceptor and NH4Cl as the nitrogen source. Hydrogen uptake activity (O2-dependent H3H oxidation) was expressed in the presence and absence of 5% H2; it reached its maximum in late logarithmic phase as the malate became limiting. This activity was very stable in stationary phase, even in the absence of exogenous H2, compared with microaerobically grown cultures; this supports the hypothesis that the exclusion of O2 is critical for maintaining the integrity of the H2 uptake system in this organism. Oxygen, as well as methylene blue and N2O, supported H2 uptake, indicating the presence of electron transport components leading to O2 in anaerobically grown A. brasilense. Nitrite (0.5 mM) inhibited H2 uptake. In cultures grown with NO3- as the terminal electron acceptor and NH4Cl as the nitrogen source, in the presence and absence of exogenous H2, only low H2 uptake activity was observed. Methylene blue, O2, N2O, NO3-, and NO2- were all capable of acting as the electron acceptor for H2 oxidation. Nitrite (0.5 mM) did not inhibit H2 uptake in NO3--grown cells, as it did in N2O-grown cells. A. brasilense appears to be one of the few organisms capable of expressing the H2 uptake system under denitrifying conditions in the absence of molecular H2.     

Relation of aerobiosis and ionic strength to the uptake of dihydrostreptomycin in Escherichia coli

Aminoglycoside antibiotics exhibit a markedly reduced antibacterial activity under anaerobic conditions. Anaerobiosis or inhibitors of electron transport produced an extensive decrease in the uptake of dihydrostreptomycin in Escherichia coli K-12. Uptake of proline or putrescine were only slightly impaired under anaerobic conditions in the presence of glucose. Both the susceptibility to and the uptake of dihydrostreptomycin under anaerobic conditions were partially restored by addition of the alternative electron acceptor, nitrate. This stimulation required functional nitrate reductase activity. Abolition of uptake by 2,4-dinitrophenol under both aerobic and anaerobic conditions indicates that streptomycin uptake requires electron transport as well as a sufficient membrane potential. In addition, the initial rate of dihydrostreptomycin uptake was competitively and reversibly inhibited by added salts. The inhibition was relatively nonspecific with respect to the identity of salt added, being approximately dependent on the ionic strength. Although dihydrostreptomycin and polyamines mutually inhibited each other's uptake, several conditions (polyamine limitation, streptomycin uptake-deficient mutants) were found in which uptake of these two substrates was oppositely affected. Aminoglycosides thus do not appear to enter on one of the usual cellular transport systems, but perhaps utilize a component of the electron transport system.     

Relationship between nitrite reduction and active phosphate uptake in the phosphate-accumulating denitrifier Pseudomonas sp. strain JR 12

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.     

Photophosphorylation Associated with Photosystem II: III. Characterization of Uncoupling, Energy Transfer Inhibition, and Proton Uptake Reactions Associated with Photosystem II Cyclic Photophosphorylation

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N′,N′-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.     

吸收胞外电子的电活性微生物

可吸收胞外电子的电活性微生物(Electroactive microorganisms,EAMs)可利用胞外固态载体的电子将二氧化碳或其他氧化态物质还原成胞外有机物、还原态无机物或自身生命活动所需的有机物。该类EAMs的出现拓宽了人们对微生物多样性的认识,在生物质能合成、污染物治理与化学物质检测等方面具有重要的应用价值。本文介绍了代表性的可吸收胞外电子EAMs的物质转化与电能转化率等基本特性,重点阐述该类EAMs基于膜蛋白的直接吸收电子机制,及基于电子穿梭体的间接吸收电子机制,提出了其在微生物电合成系统与微生物传感器中的应用前景,并从EAMs机理研究、生物膜微观机制及工程应用的角度展望其今后的研究方向。     

Trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735: Na+-stimulated anaerobic transport in cells and membrane vesicles.

Alteromonas putrefaciens NCMB 1735 required the presence of NaCl for anaerobic growth with serine, cysteine, and formate as substrate and trimethylamine oxide ( TMAO ) as external electron acceptor. When lactate was substrate, the organism grew equally well in the absence of NaCl. Anaerobic uptake of glutamate, aspartate, serine, cysteine, and lactate in resting cells was strongly stimulated with NaCl, and cytoplasmic membrane vesicles energized by electron transfer from formate to TMAO displayed active Na+-dependent uptake of serine. The data suggested that participation in transport processes was the only vital function of Na+ in A. putrefaciens. Formate- and TMAO -dependent anaerobic serine uptake in vesicles was sensitive to the protonophore carbonyl cyanide m-chlorophenyl-hydrazone and the ionophores valinomycin and gramicidin. Transport-active vesicles contained cytochromes of b and c type, and both serine uptake and TMAO reduction with formate were inhibited with the electron transfer inhibitor 2-heptyl-4-hydroxyquinoline N-oxide. Thus, reduction of TMAO to trimethylamine in A. putrefaciens appeared to be coupled with a chemiosmotic mechanism of energy conversion.     

Energy Transduction by Anaerobic Ferric Iron Respiration in Thiobacillus ferrooxidans

Formate-grown cells of the obligately chemolithoautotrophic acidophile Thiobacillus ferrooxidans were capable of formate- and elemental sulfur-dependent reduction of ferric iron under anaerobic conditions. Under aerobic conditions, both oxygen and ferric iron could be simultaneously used as electron acceptors. To investigate whether anaerobic ferric iron respiration by T. ferrooxidans is an energy-transducing process, uptake of amino acids was studied. Glycine uptake by starved cells did not occur in the absence of an electron donor, neither under aerobic conditions nor under anaerobic conditions. Uptake of glycine could be driven by formate- and ferrous iron-dependent oxygen uptake. Under anaerobic conditions, ferric iron respiration with the electron donors formate and elemental sulfur could energize glycine uptake. Glycine uptake was inhibited by the uncoupler 2,4-dinitrophenol. The results indicate that anaerobic ferric iron respiration can contribute to the energy budget of T. ferrooxidans.     

Cyanide-insensitive oxidation of ascorbate + NNN''N''-tetramethyl-p-phenylenediamine mixture by mung-bean (Phaseolus aureus) mitochondria. An energy-linked function.

Freshly prepared washed or purified mung-bean (Phaseolus aureus) mitochondria utilize oxygen with ascorbate/tetramethyl-p-phenylenediamine mixture as electron donor in the presence of KCN. ATP control of the oxygen uptake can be observed with very fresh mitochondria. The electron flow, which is inhibited by antimycin A, salicylhydroxamic acid or octylguanidine, takes place by reversed electron transport through phosphorylation site II and thence to oxygen through the cyanide-insensitive pathway. Oligomycin and low concentrations of uncoupler partially inhibit the oxygen uptake in a manner similar to that observed for other energy-linked functions of plant mitochondria. An antimycin A-insensitive oxygen uptake occurs if high concentrations of uncoupler are used, indicating that the pathway of electron flow has been altered. The process of cyanide-insensitive ascorbate oxidation is self-starting, and, since it occurs in the presence of oligomycin, it is concluded that the reaction can be energized by a single energy-conservation site associated with the cyanide-insensitive oxidase pathway.     

Passive entry of CO2 and its energy-dependent intracellular conversion to HCO3- in cyanobacteria are driven by a photosystem I-generated deltamuH+

CO(2) entry into Synechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO(2) uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO3(-). Dependence of CO(2) uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO(2) uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a DeltamuH(+) generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO(2) uptake. Under low light intensity, the rate of CO(2) uptake by a high-CO(2)-requiring mutant of Synechococcus sp. PCC7942, at a CO(2) concentration below its threshold for CO(2) fixation, was higher than that of the wild type. At saturating light intensity, net CO(2) uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO(2) to HCO3(-). These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO(2) to HCO3(-).     

Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens.

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.     

Hydrogenase in pea root nodule bacteriods

Summary Hydrogen uptake has been shown to occur with pea root nodule breis and this uptake has been shown to be confined to the bacteriods. The uptake of hydrogen by washed bacteriods, in the absence of any added substrates, has been shown to be accompanied by oxygen uptake and the ratio of hydrogen uptake to oxygen uptake in these preparations has been found to be 2:1. Substrates, provided to washed bacteriods, inhibit the uptake of hydrogen and it has been found that the utilisation of substrates, as measured by carbon dioxide evolution, is inhibited by hydrogen. It is suggested that hydrogen and substrates compete for electron carriers and that electrons from the hydrogen reduce components of the electron transport pathway and ATP is produced.The action of hydrogen on nitrogen fixation in nodule breis and washed bacterioid preparations was examined and the evidence shows that some non-competitive inhibition of nitrogen fixation, by hydrogen, occurs.     

Pathways of hydrogen uptake in the cyanobacterium Nostoc muscorum

Two pathways of hydrogen uptake in Nostoc muscorum are apparent using either oxygen or nitrogen as electron acceptor. Hydrogen uptake (under argon with some oxygen as electron acceptor assayed in the dark; oxyhydrogen reaction) is found to be more active in dense, light-limited cultures than in thin cultures when light is not limiting. Addition of bicarbonate inhibits this hydrogen uptake, because photosynthesis is stimulated. In a cell-free hydrogenase assay, a 10-fold increase of the activity can be measured, after the cells having been kept under lightlimiting conditions. After incubation under light-saturating conditions, no hydrogen uptake is found, when filaments are assayed under argon plus some oxygen. Assaying these cells under a nitrogen atmosphere, a strong hydrogen uptake occurs. The corresponding cell-free hydrogenase assay exhibits low hydrogenase activity. Furthermore, the hydrogen uptake by intact filaments under nitrogen in the light apparently is correlated with nitrogenase activity. These studies give evidence that, under certain physiological conditions, hydrogen uptake of heterocysts proceeds directly via nitrogenase, with no hydrogenase involved.Abbreviations Chl chlorophyll - DCMU (diuron) 3-3,4-dichlorophenyl)-1,1-dimethylurea - pev packed cell volume     

Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.     

Microbial photoelectrosynthesis: Feeding purple phototrophic bacteria electricity to produce bacterial biomass

Purple phototrophic bacteria are one of the main actors in chemolithotrophic carbon fixation and, therefore, fundamental in the biogeochemical cycle. These microbes are capable of using insoluble electron donors such as ferrous minerals or even carbon-based electrodes. Carbon fixation through extracellular electron uptake places purple phototrophic bacteria in the field of microbial electrosynthesis as key carbon capturing microorganisms. In this work we demonstrate biomass production dominated by purple phototrophic bacteria with a cathode (−0.6 V vs. Ag/AgCl) as electron donor. In addition, we compared the growth and microbial population structure with ferrous iron as the electron donor. We detect interaction between the cathode and the consortium showing a midpoint potential of 0.05 V (vs. Ag/AgCl). Microbial community analyses revealed different microbial communities depending on the electron donor, indicating different metabolic interactions. Electrochemical measurements together with population analyses point to Rhodopseudomonas genus as the key genus in the extracellular electron uptake. Furthermore, the genera Azospira and Azospirillum could play a role in the photoelectrotrophic consortium.     

Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis

Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<−414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.     

The mechanism of quinonediimine acceptor activity in photosynthetic electron transport.

The rates of electron flow catalyzed by a variety of unsubstituted and C- or N-methylated quinonediimine electron acceptors in a reaction requiring photosystem II in KCN-inhibited chloroplasts vary according to the structure of acceptor used. Quinonediimine, but not quinone, electron acceptor activities are inhibited by a variety of uncouplers. Kinetic analysis of this inhibition shows that it is competitive. Low concentrations of aniline also inhibit the activity of C-methylated quinonediimines, but this appears to be due to a chemical reaction between the acceptor and aniline at low pH inside the chloroplast. Light-induced uptake of a quinonediimine, p-phenylenediimine, was shown to occur in a DCMU-sensitive reaction. Methylamine uncoupling inhibits this uptake to the same extent as it inhibits electron flow. Experiments with a lipophobic acceptor, N,N,N',N'-tetramethyl-p-phenylenediimine, indicate that it catalyzes electron flow by the same mechanism as other quinonediimines. A model is proposed to account for quinonediimine-catalyzed electron flow.     

In silico analysis of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC 824 metabolic response to an external electron supply

The biological production of butanol has become an important research field and thanks to genome sequencing and annotation; genome-scale metabolic reconstructions have been developed for several Clostridium species. This work makes use of the iCAC490 model of Clostridium acetobutylicum ATCC 824 to analyze its metabolic capabilities and response to an external electron supply through a constraint-based approach using the Constraint-Based Reconstruction Analysis Toolbox. Several analyses were conducted, which included sensitivity, production envelope, and phenotypic phase planes. The model showed that the use of an external electron supply, which acts as co-reducing agent along with glucose-derived reducing power (electrofermentation), results in an increase in the butanol-specific productivity. However, a proportional increase in the butyrate uptake flux is required. Besides, the uptake of external butyrate leads to the coupling of butanol production and growth, which coincides with results reported in literature. Phenotypic phase planes showed that the reducing capacity becomes more limiting for growth at high butyrate uptake fluxes. An electron uptake flux allows the metabolism to reach the growth optimality line. Although the maximum butanol flux does not coincide with the growth optimality line, a butyrate uptake combined with an electron uptake flux would result in an increased butanol volumetric productivity, being a potential strategy to optimize the production of butanol by C. acetobutylicum ATCC 824.     

Characterization of endocytic compartments after holo-high density lipoprotein particle uptake in HepG2 cells

Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa⁵⁶⁸, used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1–2 h of continuous uptake, and a clearance 1–2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa⁵⁶⁸-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.     

Identification of an uptake hydrogenase for hydrogen-dependent dissimilatory azoreduction by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12, a representative dissimilatory azo-reducing bacterium of Shewanella genus, can grow by coupling the oxidation of hydrogen to the reduction of azo compounds as the sole electron acceptor, indicating that an uptake hydrogenase is an important component for electron transfer for azoreduction. For searching to the uptake hydrogenase in the genome of S. decolorationis, two operons, hyd and hya, were cloned and sequenced, which encode periplasmically oriented Fe-only hydrogenase and a Ni-Fe hydrogenase, respectively, according to the homologous comparison with other bacterial hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent azoreduction and growth, hyd- and hya-deficient mutants were generated by gene replacement. Hya was found to be required for hydrogen-dependent reduction of azo compound by resting cell suspensions and to be essential for growth with hydrogen as electron donor and azo compound as electron acceptor. Hyd, in contrast, was not. These findings suggest that Hya is an essential respiratory hydrogenase of dissimilatory azoreduction in S. decolorationis.