Methanol and methylamine utilization result from mutational events in Thiosphaera pantotropha

With choline as carbon source Thiosphaera pantotropha GB17 grew with a doubling time (t_d) of 6 h. The cellular yield was 55.8 g dry cell weight per mol of choline, indicating that its methyl moieties were used for growth. However, T. pantotropha was unable to grow with methanol or with methylamine as carbon source. Mutants were isolated from liquid or from solid media able to grow with methanol (Mox⁺) as carbon or methylamine as nitrogen source (Mam⁺). The Mox⁺ mutant GB17M grew with a mean t_d of 11.7h and a growth yield of 8.9 g dry cell weight per mol of methanol. Diauxic growth of strain GB17M was observed with mixtures of pyruvate and methanol as substrates in batch culture. Methanol led to the formation of methanol dehydrogenase, formate dehydrogenase, ribulosebisphosphate carboxylase and of a soluble cytochrome c-551.5. Tn5-insertional mutants defective in the thiosulfate oxidizing enzyme system or in hydrogenase acquired the Mox⁺ phenotype. However, Tn5-insertional mutants defective in either a c-type cytochrome or the molybdenum cofactor did not mutate to the Mox⁺ phenotype, indicating common functions in thiosulfate and in methanol metabolism.     

Methylamine Utilization via the N-Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C1 Assimilation and Dissimilation Pathways

Methylotrophs grow on reduced single-carbon compounds like methylamine as the sole source of carbon and energy. In Methylobacterium extorquens AM1, the best-studied aerobic methylotroph, a periplasmic methylamine dehydrogenase that catalyzes the primary oxidation of methylamine to formaldehyde has been examined in great detail. However, recent metagenomic data from natural ecosystems are revealing the abundance and importance of lesser-known routes, such as the N-methylglutamate pathway, for methylamine oxidation. In this study, we used M. extorquens PA1, a strain that is closely related to M. extorquens AM1 but is lacking methylamine dehydrogenase, to dissect the genetics and physiology of the ecologically relevant N-methylglutamate pathway for methylamine oxidation. Phenotypic analyses of mutants with null mutations in genes encoding enzymes of the N-methylglutamate pathway suggested that γ-glutamylmethylamide synthetase is essential for growth on methylamine as a carbon source but not as a nitrogen source. Furthermore, analysis of M. extorquens PA1 mutants with defects in methylotrophy-specific dissimilatory and assimilatory modules suggested that methylamine use via the N-methylglutamate pathway requires the tetrahydromethanopterin (H₄MPT)-dependent formaldehyde oxidation pathway but not a complete tetrahydrofolate (H₄F)-dependent formate assimilation pathway. Additionally, we present genetic evidence that formaldehyde-activating enzyme (FAE) homologs might be involved in methylotrophy. Null mutants of FAE and homologs revealed that FAE and FAE2 influence the growth rate and FAE3 influences the yield during the growth of M. extorquens PA1 on methylamine.     

Identification of a fourth formate dehydrogenase in Methylobacterium extorquens AM1 and confirmation of the essential role of formate oxidation in methylotrophy

A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [¹³C]formate, as well as ¹⁴C-labeling experiments, demonstrated production of labeled CO₂ in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO₂ formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.     

Bacterial yields on methanol,methylamine, formaldehyde,and formate

Several bacteria utilizing C₁-compounds as sole carbon sources were grown on these substrates in continuous culture. The molar yield values (g of cell dry wt/mol of substrate utilized) of bacteria which utilize C₁-compounds via the ribulose monophosphate pathway were between 15.7 to 17.3 when grown on methanol; while the molar yield values of bacteria which use the serine pathway for the assimilation of C₁-compounds varied between 9.8 and 13.1. The molar yield values of different bacteria which use the serine pathway decreased as the oxidation levels of the C₁-growth substrates increased. On formaldehyde the values were between 7.2 to 9.6, whereas on formate the values varied from 3.3 to 6.9. It appears that bacteria utilize C_l-compounds more efficiently via the ribulose monophosphate pathway than via the serine pathway. The oxidation step from methanol to formaldehyde (and from methylamine to formaldehyde) in the bacteria studied may be energy yielding. A comparison has been made between the experimental yield values obtained and theoretical values.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Occurrence of Methanogenic Archaea in Highly Polluted Sediments of Tropical Santos–São Vicente Estuary (São Paulo, Brazil)

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos–São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30°C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH₄/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.     

Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

Pathways leading to and from serine during growth of Pseudomonas AM1 in C1 compounds or succinate

1. The following enzymes of the phosphorylated pathway of serine biosynthesis have been found in methanol- and succinate-grown Pseudomonas AM1: phosphoglycerate dehydrogenase, phosphoserine-alpha-oxoglutarate aminotransferase and phosphoserine phosphohydrolase. Their specific activities were similar in the organism grown on either substrate. 2. A procedure for preparation of auxotrophic mutants of Pseudomonas AM1 is described involving N-methyl-N'-nitro-N-nitrosoguanidine as mutagen and a penicillin enrichment step. 3. A mutant, M-15A, has been isolated that is unable to grow on methanol and that lacks phenazine methosulphate-linked methanol dehydrogenase. The mutant is able to grow on methylamine, showing that the amine is not oxidized by way of methanol. 4. Loss of methanol dehydrogenase activity in mutant M-15A led to loss of phenazine methosulphate-linked formaldehyde dehydrogenase activity showing that the same enzyme is probably responsible for both activities. 5. A mutant, 20B-L, has been isolated that cannot grow on any C(1) compound tested but can grow on succinate. 6. Mutant 20B-L lacks hydroxypyruvate reductase, and revertants that regained the ability to grow on methanol, methylamine and formate contained hydroxypyruvate reductase activity at specific activities similar to that of the wild-type organism. This shows that hydroxypyruvate reductase is necessary for growth on methanol, methylamine and formate but not for growth on succinate. 7. The results suggest that during growth of Pseudomonas AM1 on C(1) compounds, serine is converted into 3-phosphoglycerate by a non-phosphorylated pathway, whereas during growth on succinate, phosphoglycerate is converted into serine by a phosphorylated pathway.     

Two-component system that regulates methanol and formaldehyde oxidation in Paracoccus denitrificans

A chromosomal region encoding a two-component regulatory system, FlhRS, has been isolated from Paracoccus denitrificans. FlhRS-deficient mutants were unable to grow on methanol, methylamine, or choline as the carbon and energy source. Expression of the gene encoding glutathione-dependent formaldehyde dehydrogenase (fhlA) was undetectable in the mutant, and expression of the S-formylglutathione hydrolase gene (fghA) was reduced in the mutant background. In addition, methanol dehydrogenase was immunologically undetectable in cell extracts of FhlRS mutants. These results indicate that the FlhRS sensor-regulator pair is involved in the regulation of formaldehyde, methanol, and methylamine oxidation. The effect that the FlhRS proteins exert on the regulation of C₁ metabolism might be essential to maintain the internal concentration of formaldehyde below toxic levels.     

Growth the Wolinella succinogenes on H2S plus fumarate and on formate plus sulfur as energy sources

Wolinella succinogenes was found to grow on H₂S plus fumarate with the formation of elemental sulfur and succinate. The growth rate was 0.18 h^-1 (t _d=3.8 h) and the growth yield was estimated to be 6.0 g per mol fumarate used. Growth also occurred on formate plus elemental sulfur; the products formed were H₂S and CO₂. The growth rate and estimated growth yield were 0.58 h^-1 (t _d=1.2 h) and 3.5 g per mol formate used, respectively. These results suggest that certain chemotrophic anaerobes may be involved in both the formation and reduction of sulfur.     

A novel plant-associated thermotolerant alkaliphilic methylotroph of the genusParacoccus

Strain GB isolated from the maize rhizosphere is a gram-negative, aerobic, non-spore-forming, nonpigmented, nonmotile, chemolithotrophic, facultatively methylotrophic bacterium. Cells are cocci or short rods. The strain does not require vitamins. Optimum growth in a medium with methanol occurs at 38–42°C at pH 8.0–9.2. The doubling time is 12 h. In addition to methanol, the bacterium can grow on methylamine, dimethylformamide, acetone, thiosulfate + NaHCO₃, and in an atmosphere of H₂ + CO₂ + O₂. Methanol and methylamine are oxidized by the respective dehydrogenases to CO₂ via formaldehyde and formate, respectively. The CO₂ produced is assimilated via the ribulose bisphosphate pathway. Fatty acids are dominated by cyclopropanoic (58–61%), palmitic (24–26%), and octadecanoic (8–9%) acids. The main phospholipids are phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The major ubiquinone is Q₁₀. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The culture liquid exhibits cytokinin activity. The G+C content of DNA is 62.5 mol %, as determined from the DNA thermal denaturation temperature T_m). Strain GB shows a moderate degree of DNA-DNA homology (<40%) with the type representatives of the genusParacoccus. Based on the data obtained, the bacterium was classified as a new species of this genus, namedP. kondratievae.     

Microbial metabolism of pyridinium compounds. Metabolism of 4-carboxy-1-methylpyridinium chloride, a photolytic product of paraquat

1. A bacterium, Achromobacter D, isolated from garden soil by elective culture, utilized N-methylisonicotinic acid (4-carboxy-1-methylpyridinium chloride) as sole carbon source. 2. Extracts of N-methylisonicotinate-grown cells oxidized this substrate only after supplementation with a source of nicotinamide nucleotides and then consumed 1 mol of O₂ and released 1 mol of CO₂/mol of N-methylisonicotinate supplied. 3. The N-methyl group of the substrate was released as methylamine whereas the five C atoms of the pyridine ring were accounted for as succinate and formate. The CO₂ evolved by extracts was believed to derive from the carboxyl group on C-4 of the heterocyclic ring. 4. The immediate precursor of the succinate end-product was succinic semialdehyde; the inducible nature of succinic semialdehyde dehydrogenase in N-methylisonicotinate-grown cells supported this finding. 5. There was no evidence for monohydroxylation of the ring, but the time sequence of the appearance of the end-products indicated that the oxygen-requiring, NADH-requiring and decarboxylation steps clearly preceded the formation of methylamine and succinate. 6. The results are consistent with the oxidative cleavage of a partially reduced heterocyclic ring followed by several hydrolytic and dehydrogenase steps resulting in the appearance of the end-products.     

Metabolism of Formate in Methanobacterium formicicum

Methanobacterium formicicum strain JF-1 was cultured with formate as the sole energy source in a pH-stat fermentor. Growth was exponential, and both methane production and formate consumption were linear functions of the growth rate. Hydrogen was produced in only trace amounts, and the dissolved H₂ concentration of the culture medium was below 1 μM. The effect of temperature or pH on the rate of methane formation was studied with a single fermentor culture in mid-log phase that was grown with formate under standard conditions at 37°C and pH 7.6. Methane formation from formate occurred over the pH range from 6.5 to 8.6, with a maximum at pH 8.0. The maximum temperature of methanogenesis was 56°C. H₂ production increased at higher temperatures. Hydrogen and formate were consumed throughout growth when both were present in saturating concentrations. The molar growth yields were 1.2 ± 0.06 g (dry weight) per mol of formate and 4.8 ± 0.24 g (dry weight) per mol of methane. Characteristics were compared for cultures grown with either formate or H₂-CO₂ as the sole energy source at 37°C and pH 7.6; the molar growth yield for methane of formate cultures was 4.8 g (dry weight) per mol, and that of H₂-CO₂ cultures was 3.5 g (dry weight) per mol. Both formate and H₂-CO₂ cultures had low efficiencies of electron transport phosphorylation; formate-cultured cells had greater specific activities of coenzyme F₄₂₀ than did H₂-CO₂-grown cultures. Hydrogenase, formate dehydrogenase, chromophoric factor F₃₄₂, and low levels of formyltetrahydrofolate synthetase were present in cells cultured with either substrate. Methyl viologen-dependent formate dehydrogenase was found in the soluble fraction from broken cells.     

Mutagenesis of the C1 oxidation pathway in Methanosarcina barkeri: new insights into the Mtr/Mer bypass pathway

A series of Methanosarcina barkeri mutants lacking the genes encoding the enzymes involved in the C1 oxidation/reduction pathway were constructed. Mutants lacking the methyl-tetrahydromethanopterin (H₄MPT):coenzyme M (CoM) methyltransferase-encoding operon (Δmtr), the methylene-H₄MPT reductase-encoding gene (Δmer), the methylene-H₄MPT dehydrogenase-encoding gene (Δmtd), and the formyl-methanofuran:H₄MPT formyl-transferase-encoding gene (Δftr) all failed to grow using either methanol or H₂/CO₂ as a growth substrate, indicating that there is an absolute requirement for the C1 oxidation/reduction pathway for hydrogenotrophic and methylotrophic methanogenesis. The mutants also failed to grow on acetate, and we suggest that this was due to an inability to generate the reducing equivalents needed for biosynthetic reactions. Despite their lack of growth on methanol, the Δmtr and Δmer mutants were capable of producing methane from this substrate, whereas the Δmtd and Δftr mutants were not. Thus, there is an Mtr/Mer bypass pathway that allows oxidation of methanol to the level of methylene-H₄MPT in M. barkeri. The data further suggested that formaldehyde may be an intermediate in this bypass; however, no methanol dehydrogenase activity was found in Δmtr cell extracts, nor was there an obligate role for the formaldehyde-activating enzyme (Fae), which has been shown to catalyze the condensation of formaldehyde and H₄MPT in vitro. Both the Δmer and Δmtr mutants were able to grow on a combination of methanol plus acetate, but they did so by metabolic pathways that are clearly distinct from each other and from previously characterized methanogenic pathways.     

The linear oxidation of formaldehyde to CO2 as the proper energy generating sequence for the assimilation of methanol in Acetobacter methanolicus MB58

Acetobacter methanolicus MB58 can grow on methanol. Since this substrate exhibits to be energy deficient there must be a chance to oxidize methanol to CO₂ merely for purpose of energy generation. For the assimilation of methanol the FBP variant of the RuMP pathway is used. Hence methanol can be oxidized cyclically via 6-phosphogluconate. Since Acetobacter methanolicus MB58 possesses all enzymes for a linear oxidation via formate the question arises which of both sequences is responsible for generation of the energy required. In order to clarify this the linear sequence was blocked by inhibiting the formate dehydrogenase with hypophosphite and by mutagenesis inducing mutants defective in formaldehyde or formate dehydrogenase. It has been shown that the linear dissimilatory sequence is indispensable for methylotrophic growth. Although the cyclic oxidation of formaldehyde to CO₂ has not been influenced by hypophosphite and with mutants both the wild type and the formaldehyde dehydrogenase defect mutants cannot grown on methanol. The cyclic oxidation of formaldehyde does not seem to be coupled to a sufficient energy generation, probably it operates only detoxifying and provides reducing equivalents for syntheses. The regulation between assimilation and dissimilation of formaldehyde in Acetobacter methanolicus MB58 is discussed.Abbreviations ATP Adenosine-5-triphosphate - DCPIP 2,6-dichlorphenolindophenol - DW dry weight - ETP electron transport phosphorylation - FBP fructose-1,6-bisphosphate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PMS phenazine methosulfate - RuMP ribulose monophosphate - Ru5P ribulose-5-phosphate - SDS sodiumdodecylsulphate - TCA tricarboxylic acid - TYB toluylene blue Dedicated to Prof. Dr. Dr. S. M. Rapoport on occasion of his 75th birthday     

The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B12- and Tetrahydrofolate-Dependent Chloromethane Metabolism in Methylobacterium extorquens CM4: A Proteomic and Bioinformatics Study

Chloromethane (CH₃Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B₁₂ are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B₁₂-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.     

Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.     

Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline.

The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.     

Growth with hydrogen,and further physiological characteristics of Desulfotomaculum species

Growth of Desulfotomaculum orientis, D. ruminis, D. nigrificans and the Desulfotomaculum strains TEP, TWC and TWP, that were newly isolated with sulfate and fatty acids, was studied using defined mineral media. Four of these strains grew with hydrogen plus sulfate as the only energy source. Under these conditions the growth yield of D. orientis in batch culture was 7.5 g cell dry mass per mol sulfate reduced. Growth on methanol with growth yields of about 6 g cell dry mass per mol sulfate was obtained with D. orientis and strain TEP. All strains tested grew slowly with formate as electron donor. Fatty acids from propionate to palmitate were utilized by the strains TEP, TWC and TWP. D. orientis and the strains TEP and TWC were able to utilize the methoxyl groups of trimethoxybenzoate for growth. D. orientis was found to grow chemoautotrophically with hydrogen, carbon dioxide and sulfate; during growth with C₁-compounds no additional organic carbon source was required. Furthermore, D. orientis was able to grow slowly in sulfate-free medium with formate, methanol, ethanol lactate, pyruvate or trimethoxybenzoate. Under these conditions acetate was excreted, indicating the function of carbon dioxide as electron acceptor in a homoacetogenic process. A growth-promoting effect of pyrophosphate added to the medium of Desulfotomaculum species was not observed. The results show a high catabolic and anabolic versatility among Desulfotomaculum species, and indicate that electron transport to sulfate can be the sole energy conserving process in this genus.