Characterization of the H2- and CO-dependent chemolithotrophic potentials of the acetogens Clostridium thermoaceticum and Acetogenium kivui.

Strains of Clostridium thermoaceticum were tested for H2- and CO-dependent growth in a defined medium containing metals, minerals, vitamins, cysteine-sulfide, CO2-bicarbonate, and H2 or CO. Ten of the thirteen strains tested grew at the expense of H2 and CO, and C. thermoaceticum ATCC 39073 was chosen for further study. The doubling times for H2- and CO-dependent growth under chemolithotrophic conditions (the defined medium with nicotinic acid as sole essential vitamin and sulfide as sole reducer) were 25 and 10 h, respectively. Product stiochiometries for chemolithotrophic cultures approximated: 4.1H2 + 2.4CO2----CH3COOH + 0.1 cell C + 0.3 unrecovered C and 6.8CO----CH3COOH + 3.5CO2 + 0.4 cell C + 0.9 unrecovered C. H2-dependent growth produced significantly higher acetate concentrations per unit of biomass synthesized than did CO- or glucose-dependent growth. In contrast, the doubling time for H2-dependent growth under chemolithotrophic conditions (the defined medium without vitamins and sulfide as sole reducer) by Acetogenium kivui ATCC 33488 was 2.7 h; as a sole energy source, CO was not growth supportive for A. kivui. The YH2 values for A. kivui and C. thermoaceticum were 0.91 and 0.46 g of cell dry weight per mol of H2 consumed, respectively; the YCO value for C. thermoaceticum was 1.28 g of cell dry weight per mol of CO consumed. The specific activities of hydrogenase and CO dehydrogenase in both acetogens were influenced by the energy source utilized for growth and were significantly lower in C. thermoaceticum than in A. kivui. With extracts of H2-cultivated cells and benzyl viologen as electron acceptor, the Vmax values for hydrogenase from C. thermoaceticum and A. kivui were 155.7 and 1,670 micromoles of H2 oxidized per min mg of protein, respectively; the Vmax values for CO dehydrogenase from C. thermoaceticum and A. kivui were 90.6 and 2,973 micromoles of CO oxidized per min per mg of protein, respectively.     

Carbon monoxide-dependent methyl coenzyme M methylreductase in acetotrophic Methosarcina spp

Cell extracts of acetate-grown Methanosarcina strain TM-1 and Methanosarcina acetivorans both contained CH3-S-CoM methylreductase activity. The methylreductase activity was supported by CO and H2 but not by formate as electron donors. The CO-dependent activity was equivalent to the H2-dependent activity in strain TM-1 and was fivefold higher than the H2-dependent activity of M. acetivorans. When strain TM-1 was cultured on methanol, the CO-dependent activity was reduced to 5% of the activity in acetate-grown cells. Methanobacterium formicicum grown on H2-CO2 contained no CO-dependent methylreductase activity. The CO-dependent methylreductase of strain TM-1 had a pH optimum of 5.5 and a temperature optimum of 60 degrees C. The activity was stimulated by the addition of MgCl2 and ATP. Both acetate-grown strain TM-1 and acetate-grown M. acetivorans contained CO dehydrogenase activities of 9.1 and 3.8 U/mg, respectively, when assayed with methyl viologen. The CO dehydrogenase of acetate-grown cells rapidly reduced FMN and FAD, but coenzyme F420 and NADP+ were poor electron acceptors. No formate dehydrogenase was detected in either organism when grown on acetate. The results suggest that a CO-dependent CH3-S-CoM methylreductase system is involved in the pathway of the conversion of acetate to methane and that free formate is not an intermediate in the pathway.     

Carbon monoxide-dependent growth of Rhodospirillum rubrum.

Under dark, anaerobic conditions in the presence of sufficient nickel, Rhodospirillum rubrum grows with a doubling time of under 5 h by coupling the oxidation of CO to the reduction of H+ to H2. CO-dependent growth of R. rubrum UR294, bearing a kanamycin resistance cassette in cooC, depends on a medium nickel level ninefold higher than that required for optimal growth of coo+ strains.     

Biotransformations of aromatic aldehydes by acetogenic bacteria

Vanillin was subject to O demethylation and supported growth of Clostridium formicoaceticum and Clostridium thermoaceticum. Vanillin was also stimulatory to the CO-dependent growth of Peptostreptococcus productus. The aldehyde substituent of vanillin was metabolized by routes which were dependent upon both the acetogen and a co-metabolizable substrate (e.g. carbon monoxide [CO]). C. formicoaceticum and C. thermoaceticum oxidized the aldehyde group of vanillin to the carboxyl level, while P. productus reduced the aldehyde group of vanillin to the alcohol level. In contrast, during CO-dependent growth, C. thermoaceticum reduced 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol while P. productus both reduced and oxidized 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol and 4-hydroxybenzoate, respectively. These metabolic potentials indicate aromatic aldehydes may affect the flow of reductant during acetogenesis.     

Ferredoxin requirement for electron transport from the carbon monoxide dehydrogenase complex to a membrane-bound hydrogenase in acetate-grown Methanosarcina thermophila

Cell extracts from acetate-grown Methanosarcina thermophila contained CO-oxidizing:H2-evolving activity 16-fold greater than extracts from methanol-grown cells. Following fractionation of cell extracts into soluble and membrane components, CO-dependent H2 evolution and CO-dependent methyl-coenzyme M methylreductase activities were only present in the soluble fraction, but addition of the membrane fraction enhanced both activities. A b-type cytochrome(s), present in the membrane fraction, was linked to a membrane-bound hydrogenase. CO-oxidizing:H2-evolving activity was reconstituted with: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The ferredoxin also coupled CO oxidation by CO dehydrogenase complex to metronidazole reduction.     

Metabolism of H2-CO2, methanol, and glucose by Butyribacterium methylotrophicum.

The fermentative metabolism of Butyribacterium methylotrophicum grown on either H2-CO2, methanol, glucose, or CO is described. The following reaction stoichiometries were obtained: 1.00 H2 + 0.52 CO2 leads to 0.22 acetate + 0.06 cell C; 1 methanol + 0.18 CO2 + 0.01 acetate leads to 0.24 butyrate + 0.29 cell C; and 1.00 glucose leads to 0.31 CO2 + 1.59 acetate + 0.21 butyrate + 0.13 H2 + 1.58 cell C. Cell yields of 1.7 g (dry weight) per mol of H2, 8.2 g (dry weight) per mol of methanol, 42.7 g (dry weight) per mol of glucose, and 3.0 g (dry weight) per mol of CO were obtained from linear plots of cell synthesis and substrate consumption. Doubling times of 9.0, 9.0, and 3 to 4 h were observed during batch growth on H2-CO2, methanol, and glucose, respectively. Indicative of a growth factor limitation, glucose fermentation in defined medium displayed a lower cell synthesis efficiency than when yeast extract (0.05%) was present. B. methylotrophicum fermentation displayed atypically high substrate/cell carbon synthesis conversion ratios for an anaerobe, as greater than 24% of the carbon was assimilated into cells during growth on methanol or glucose. The data indicate that B. methylotrophicum conserves carbon-bound electrons during growth on single-carbon or multicarbon substrates.     

Characterization of a CO-dependent O-demethylating enzyme system from the acetogen Clostridium thermoaceticum.

An inducible O-demethylating enzyme system was characterized from Clostridium thermoaceticum cultivated at the expense of syringate. Glucose and methanol, but not CO, partially repressed its expression. Induced whole cells catalyzed the carbon monoxide (CO)-dependent O demethylation of methoxylated aromatic compounds with the concomitant formation of acetate. Pyruvate and, to a lesser extent, H2-CO2 could replace CO in these reactions. KCN inhibited pyruvate-dependent activity but not the CO-dependent activity. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide, the protonophore carbonyl cyanide m-chlorophenylhydrazone, and methyl viologen did not appreciably inhibit O demethylation by induced cells, whereas Triton X-100 was inhibitory. The enzyme system appeared to convert syringate sequentially to 5-hydroxyvanillate and gallate. The proposed overall reaction stoichiometry was as follows: syringate + 2CO + 2H2O----gallate + 2 acetates. Growth-supportive methoxylated aromatic compounds were O demethylated by syringate-cultivated cells and inhibitory to syringate O demethylation.     

CO metabolism of Desulfovibrio vulgaris strain Madison: physiological function in the absence or presence of exogeneous substrates

Abstract Trace amounts of carbon monoxide were produced and subsequently consumed during the growth of Desulfovibrio vulgaris on organic electron donors. D. vulgaris also utilized carbon monoxide as the sole electron donor for growth and sulfate reduction. Growth of D. vulgaris on CO, H₂ or organic electron donors was inhibited at ≥4.5% CO in the culture headspace. At lower CO concentrations, hydrogen was produced as a consequence of CO consumption and consumed when the CO partial pressure was decreased. The rate of CO consumption was ten-fold higher in D. vulgaris grown on either CO, lactate or pyruvate than when cells were grown on H₂ as electron donor. The physiological function of CO metabolism and a CO-dependent hydrogen cycle in D. vulgaris is discussed.     

Cloning and DNA homology of 3-isopropylmalate dehydrogenase genes from thermophilic bacilli

Abstract Clostridium thermoaceticum was cultivated heterotrophically under CO₂, carbon monoxide (CO), and H₂ gas phases. Formate dehydrogenase (FDH) levels increased 4-fold in CO cultures; formyltetrahydrofolate synthetase (FTS) levels were not influenced by the cultivation gas phases tested. While CO dehydrogenase (CODH) was slightly stimulated in CO cultures, CO-dependent acetyl phosphate-synthesizing system (APSS) activity increased sharply in both CO and H₂ cultures. Y _glucose values increased, whereas doubling times and acetate to biomass ratios decreased significantly in CO cultures, suggesting that CO cultures were energetically dissimilar to non-CO cultures. This finding, together with the absence of CO-dependent ATP-independent synthesis of formyltetrahydrofolate (formyl-THF), supports the hypothesis that conservation of CO-derived energy involves electron transport phosphorylation.     

Comparative evaluation of the metabolic potentials of different strains of Peptostreptococcus productus: utilization and transformation of aromatic compounds

Three strains of Peptostreptococcus productus were tested for growth at the expense of methoxylated aromatic compounds. Strain M8A-18 (human fecal isolate) was unable to utilize methoxylated aromatic compounds. While the type strain ATCC 27340 (human septicemia isolate) was capable of minimal growth with methoxylated aromatic compounds, ATCC 35244 (sewage sludge isolate) displayed significant growth on methoxylated aromatic compounds. Methoxylated phenols, benzoates, benzyl alcohol and phenylacrylates supported the growth of ATCC 35244 and were O-demethylated to their respective hydroxylated derivatives. During O-methyl- or CO-dependent growth, the double bond of the acrylate side chain of certain methoxylated and non-methoxylated phenylacrylates was reduced. Although other aromatic substituent groups (-COOH and -CH3) were transformed during CO-dependent growth, in short-term growth studies, the aromatic ring was not subject to reduction or degradation. Of the three strains tested, only strain M8A-18 failed to grow at the expense of carbon monoxide (CO).     

Oxidation of carbon monoxide and methane by Pseudomonas methanica.

The oxidation of carbon monoxide and methane by suspensions and ultrasonic extracts of Pseudomonas methanica was studied. A continuous assay for the oxidation of CO to CO2 was devised, using O2 and CO2 electrodes in combination. Stoicheiometries of CO-dependent CO2 formation, O2 consumption and NADH oxidation, and the partial stoicheiometries of methane-dependent NADH oxidation, suggest the involvement of a mono-oxygenase in these oxidations. Evidence is presented suggesting methane and CO oxidation are catalysed by a single enzyme system, distinct, at least in part, from the NADH oxidase present in extracts. Ethanol was able to provide the reductant necessary for CO oxidation by cell suspensions, though the metabolism of ethanol by P. methanica was found unlikely to result in substrate-level formation of NADH; the means whereby alcohol oxidation could supply reductant for the mono-oxygenase are discussed.     

Carbon monoxide-dependent energy metabolism in anaerobic bacteria and archaea

Despite its toxicity for the majority of living matter on our planet, numerous microorganisms, both aerobic and anaerobic, can use carbon monoxide (CO) as a source of carbon and/or energy for growth. The capacity to employ carboxidotrophic energy metabolism anaerobically is found in phylogenetically diverse members of the Bacteria and the Archaea. The oxidation of CO is coupled to numerous respiratory processes, such as desulfurication, hydrogenogenesis, acetogenesis, and methanogenesis. Although as diverse as the organisms capable of it, any CO-dependent energy metabolism known depends on the presence of carbon monoxide dehydrogenase. This review summarizes recent insights into the CO-dependent physiology of anaerobic microorganisms with a focus on methanogenic archaea. Carboxidotrophic growth of Methanosarcina acetivorans, thought to strictly rely on the process of methanogenesis, also involves formation of methylated thiols, formate, and even acetogenesis, and, thus, exemplifies how the beneficial redox properties of CO can be exploited in unexpected ways by anaerobic microorganisms.     

Differential effects of sodium on hydrogen- and glucose-dependent growth of the acetogenic bacterium Acetogenium kivui

Acetogenium kivui could not be revived or maintained in a sodium-deficient medium (0.2 mM sodium) under H2-dependent conditions, and neither lithium nor potassium replaced the sodium requirement of H2-cultivated cells. Conversely, the revival and maintenance of glucose-cultivated cells did not display a dependency on supplemental sodium. In the absence of growth, formate became a major end product in both sodium-deficient and metabolically impaired H2-grown cultures of A. kivui. Harmaline, a putative inhibitor of Na+/H+ antiporters, uncoupled acetogenesis from H2-dependent growth but was less effective when growth was at the expense of glucose. Significantly, carbon monoxide (CO) stimulated H2-dependent growth of A. kivui but inhibited glucose-dependent growth. Collectively, these findings demonstrate that sodium plays a critical role in the H2-dependent bioenergetics of A. kivui and indicate that autotrophic and heterotrophic cells may utilize dissimilar mechanisms of energy conservation. In contrast to the growth of A. kivui, supplemental sodium was not required for the glucose-, H2-, and CO-dependent growth of Clostridium thermoaceticum.     

Differential effects of sodium on hydrogen- and glucose-dependent growth of the acetogenic bacterium Acetogenium kivui.

Acetogenium kivui could not be revived or maintained in a sodium-deficient medium (0.2 mM sodium) under H2-dependent conditions, and neither lithium nor potassium replaced the sodium requirement of H2-cultivated cells. Conversely, the revival and maintenance of glucose-cultivated cells did not display a dependency on supplemental sodium. In the absence of growth, formate became a major end product in both sodium-deficient and metabolically impaired H2-grown cultures of A. kivui. Harmaline, a putative inhibitor of Na+/H+ antiporters, uncoupled acetogenesis from H2-dependent growth but was less effective when growth was at the expense of glucose. Significantly, carbon monoxide (CO) stimulated H2-dependent growth of A. kivui but inhibited glucose-dependent growth. Collectively, these findings demonstrate that sodium plays a critical role in the H2-dependent bioenergetics of A. kivui and indicate that autotrophic and heterotrophic cells may utilize dissimilar mechanisms of energy conservation. In contrast to the growth of A. kivui, supplemental sodium was not required for the glucose-, H2-, and CO-dependent growth of Clostridium thermoaceticum.     

Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system.

A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif.     

Gas Exchange and C Allocation in Dunaliella salina Cells in Response to the N Source and CO2 Concentration Used for Growth

The halotolerant alga Dunaliella salina was cultured on 10 mM NH4+ or NO3- with air CO2 or 5% (v/v) CO2. Cells grown on NH4+ rather than NO3- were up to 17% larger in volume but had similar division rates. The photosynthetic K0.5 of dissolved inorganic C per cell was reduced, but the light- and CO2-saturated photosynthesis, dark respiration, and light-independent fixation rates were increased. The cells exhibited 2- to 5-fold greater activities of ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase and carboxykinase, and carbonic anhydrase and more soluble and ribulose-1,5-bisphosphate carboxylase/oxygenase protein. Chlorophyll and [beta]-carotene also increased by 30 to 70%. However, starch and glycerol decreased, indicating that C was reallocated from carbohydrates into protein and pigments by growth on NH4+. Algae cultured on air-CO2 rather than a high CO2 concentration were 44% smaller with 55 to 67% lower cell division rates and thus appeared C-limited, despite the operation of a CO2-concentrating mechanism. Cells cultured on air-CO2 had less protein and starch and 28% more glycerol, but the pigment content was unchanged. In only one growth regime was the cell glycerol concentration sufficient to maintain osmotic equilibrium with the external medium, indicating that an additional osmoticum was required. It appears that the N source, as well as the growth [CO2], substantially modifies photosynthetic and growth characteristics, light-independent C metabolism, and C-allocation patterns of D. salina cells.     

Anaerobic c(1) metabolism of the o-methyl-C-labeled substituent of vanillate

The O-methyl substituents of aromatic compounds constitute a C(1) growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C(1) substrate was determined by C radiotracer techniques. O-[methyl-C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C(1) substrate. The data showed that for every O-methyl carbon converted to [C]acetate, two were oxidized to CO(2). Quantitation of the carbon recovered in the two products, acetate and CO(2), indicated that acetate was formed in part by the fixation of unlabeled CO(2). The specific activity of C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO(2) and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-C]vanillate by strain TH-001 can be described as follows: 3CH(3)OC(7)H(5)O(3) + CO(2) + 4H(2)O --> CH(3)COOH + 2CO(2) + 10H + 10e + 3HOC(7)H(5)O(3).     

CO-dependent H2 evolution by Rhodospirillum rubrum: role of CODH:CooF complex

Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H(2) evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H(2) is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe(4)S(4) clusters. The observation of 2 Fe(4)S(4) clusters for CooF contradicts the prediction of 4 Fe(4)S(4) clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H(2) evolution indicates that approximately 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H(2) evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H(2) evolution activity.     

Metabolism of n-propylamine, isopropylamine, and 1,3-propane diamine by Mycobacterium convolutum.

Mycobacterium convolutum strain NPA-1 can utilize n-propylamine (NPA), isopropylamine (IPA), and 1,3-propane diamine (PD) as sole source of carbon, nitrogen, and energy. Enzyme assays, fatty acid profiles, and 14CO2 incorporation experiments indicate that NPA is deaminated to propionate and further metabolized via the methylmalonyl succinate pathway, and IPA and PD were metabolized (after deamination) through a C2 + C1 cleavage. An inducible amine dehydrogenase was present in cell extracts after growth on the three amines. Polyacrylamide gel electrophoresis of cell extracts from NPA- and IPA-grown cells yielded one major band of amine dehydrogenase activity. When extracts of NPA-grown cells were assayed with NPA, IPA, or PD as substrate, the relative position of the major band on gel electrophoresis was equivalent. Similar results were obtained with extracts prepared from IPA-grown cells. Sephadex G-100 chromatography also indicated one major peak of activity. This suggests that one enzyme of broad specificity is involved in deamination of IPA, NPA, and PD. IPA-grown cells utilized NPA readily, whereas NPA-grown cells could not utilize IPA without lag. Since amine dehydrogenase activity was present in extracts of cells after growth on either substrate, this lag was probably due to the inability to transport IPA without an induction period. The molecular weight of the amine dehydrogenase was approximately 38,500 as determined by gel filtration.