Formaldehyde activating enzyme (Fae) and hexulose-6-phosphate synthase (Hps) in <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis>: a possible function in ribose-5-phosphate biosynthesis

Formaldehyde activating enzyme (Fae) was first discovered in methylotrophic bacteria, where it is involved in the oxidation of methanol to CO₂ and in formaldehyde detoxification. The 18 kDa protein catalyzes the condensation of formaldehyde with tetrahydromethanopterin (H₄MPT) to methylene-H₄MPT. We describe here that Fae is also present and functional in the methanogenic archaeon Methanosarcina barkeri. The faeA homologue in the genome of M. barkeri was heterologously expressed in Escherichia coli and the overproduced purified protein shown to actively catalyze the condensation reaction: apparent V _max=13 U/mg protein (1 U=μmol/min); apparent Km for H₄MPT=30 μM; apparent Km for formaldehyde=0.1 mM. By Western blot analysis the concentration of Fae in cell extracts of M. barkeri was determined to be in the order of 0.1% of the soluble cell proteins. Besides the faeA gene the genome of M. barkeri harbors a second gene, faeB-hpsB, which is shown to code for a 42 kDa protein with both Fae activity (3.6 U/mg) and hexulose-6-phosphate synthase (Hps) activity (4.4 U/mg). The results support the recent proposal that in methanogenic archaea Fae and Hps could have a function in ribose phosphate synthesis.     

Formate-Dependent H2 Production by the Mesophilic Methanogen Methanococcus maripaludis

Methanococcus maripaludis, an H₂- and formate-utilizing methanogen, produced H₂ at high rates from formate. The rates and kinetics of H₂ production depended upon the growth conditions, and H₂ availability during growth was a major factor. Specific activities of resting cells grown with formate or H₂ were 0.4 to 1.4 U·mg⁻¹ (dry weight). H₂ production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (K_f) was 3.6 mM. In contrast, in H₂-grown cells this process followed sigmoidal kinetics, and the K_f was 9 mM. A key enzyme for formate-dependent H₂ production was formate dehydrogenase, Fdh. H₂ production and growth were severely reduced in a mutant containing a deletion of the gene encoding the Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H₂ production by a mutant containing a deletion of the coenzyme F₄₂₀-reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H₂ production comprised Fdh1 and Fru. Because a Δfru-Δfrc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F₄₂₀-dependent methylene-H₄MTP dehydrogenase (Mtd) or the H₂-forming methylene-H₄MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H₂ production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H₂ production was not a direct intermediate of methanogenesis. In conclusion, high rates of formate-dependent H₂ production demonstrated the potential of M. maripaludis for the microbial production of H₂ from formate.     

Effect of methanogenic substrates on coenzyme F420-dependent N5,N10-methylene-H4MPT dehydrogenase,N5,N10-methenyl-H4MPT cyclohydrolase and F420-reducing hydrogenase activities in Methanosarcina barkeri

We measured F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydro-methanopterin dehydrogenase, N⁵, N¹⁰-methenyltetrahydro-methanopterin cyclohydrolase, and F₄₂₀-reducing hydrogenase levels in Methanosarcina barkeri grown on various substrates. Variation in dehydrogenase levels during growth on a specific substrate was usually <3-fold, and much less for cyclohydrolase. H₂–CO₂-, methanol-, and H₂–CO₂+ methanol-grown cells had roughly equivalent levels of dehydrogenase and cyclohydrolase. In acetate-grown cells cyclohydrolase level was lowered 2 to 3-fold and dehydrogenase 10 to 80-fold; this was not due to repression by acetate, since, if cultures growing on acetate were supplemented with methanol or H₂–CO₂, dehydrogenase levels increased 14 to 19-fold, and cyclohydrolase levels by 3 to 4-fold. Compared to H₂–CO₂- or methanol-grown cells, acetate-or H₂–CO₂ + methanol-grown cells had lower levels of and less growth phase-dependent variation in hydrogenase activity. Our data are consistent with the following hypotheses: 1. M. barkeri oxidizes methanol via a portion of the CO₂-reduction pathway operated in the reverse direction. 2. When steps from CO₂ to CH₃-S-CoM in the CO₂-reduction pathway (in either direction) are not used for methanogenesis, hydrogenase activity is lowered.Abbreviations MF methanofuran - H₄MPT 5,6,7,8-tetrahydromethanopterin - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP heterodisulfide of HS-CoM and HS-HTP - F₄₂₀ coenzyme F₄₂₀ (a 7,8-didemethyl-8-hydroxy-5-deaza-riboflavin derivative) - H₂F₄₂₀ reduced coenzyme F₄₂₀ - HC⁺=H₄MPT N⁵,N¹⁰-methenyl-H₄MPT - H₂C=H₄MPT N⁵,N¹⁰-methylene-H₄MPT - H₃C=H₄MPT N⁵-methyl-H₄MPT - BES 2-bromoethanesulfonic acid     

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.     

Enzymes and coenzymes of the carbon monoxide dehydrogenase pathway for autotrophic CO2 fixation in Archaeoglobus lithotrophicus and the lack of carbon monoxide dehydrogenase in the heterotrophic A. profundus

Archaeoglobus lithotrophicus is a hyperthermophilic Archaeon that grows on H₂ and sulfate as energy sources and CO₂ as sole carbon source. The autotrophic sulfate reducer was shown to contain all the enzyme activities and coenzymes of the reductive carbon monoxide dehydrogenase pathway for autotrophic CO₂ fixation as operative in methanogenic Archaea. With the exception of carbon monoxide dehydrogenase these enzymes and coenzymes were also found in A. profundus. This organism grows lithotrophically on H₂ and sulfate, but differs from A. lithotrophicus in that it cannot grow autotrophically: A. profundus requires acetate and CO₂ for biosynthesis. The absence of carbon monoxide dehydrogenase in A. profundus is substantiated by the observation that this organism, in contrast to A. lithotrophicus, is not mini-methanogenic and contains only relatively low concentrations of corrinoids.Abbreviations F ₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formylmethanofuran - H ₄MPT 5,6,7,8-tetrahydromethanopterin - CHO–H ₄MPT N⁵ formyl-H₄MPT - CHH₄MPT⁺N⁵ methenyl-H₄MPT - CH ₂=H₄MPT N⁵, N¹⁰ methylene-H₄MPT - CH ₃–H₄MPT N⁵ methyl-H₄MPT - H ₄F tetrahydrofolate - I U 1 mol/min - t _d doubling time     

N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

Methylene-H₄MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F₄₂₀ as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K _m for methylene-H₄MPT of 16 M, a K _m for F₄₂₀H₂ of 4 M, and a V _max of 450 U/mg (K_cat=265 s^-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F₄₂₀ (0.2 mM), methylene-H₄MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H₄MPT reductase (F₄₂₀-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formyl-MFR - 1 U 1 mol/min     

Sulfate-Dependent Interspecies H2 Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 μmol of H₂ per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H₂ concentration was ≤2 μmol/liter. The fractions of ¹⁴CO₂ produced from [¹⁴C]methanol and 2-[¹⁴C]acetate increased from 0.26 and 0.16, respectively, in pure culture to 0.59 and 0.33, respectively, in coculture. Under these conditions, approximately 42% of the available electron equivalents derived from methanol or acetate were transferred and were utilized by D. vulgaris to reduce approximately 33 μmol of sulfate per 100 μmol of substrate consumed. As a direct consequence, methane formation in cocultures was two-thirds that observed in pure cultures. The addition of 5.0 mM sodium molybdate or exogenous H₂ decreased the effects of D. vulgaris on the metabolism of M. barkeri. An analysis of growth and carbon and electron flow patterns demonstrated that sulfate-dependent interspecies H₂ transfer from M. barkeri to D. vulgaris resulted in less methane production, increased CO₂ formation, and sulfide formation from substrates not directly utilized by the sulfate reducer as electron donors for energy metabolism and growth.     

Regulation and Physiological Role of the DAS1 Gene, Encoding Dihydroxyacetone Synthase, in the Methylotrophic Yeast Candida boidinii

The physiological role of dihydroxyacetone synthase (DHAS) in Candida boidinii was evaluated at the molecular level. The DAS1 gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that DAS1 expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The DAS1 gene was disrupted in one step in the host genome (das1Δ strain), and the growth of the das1Δ strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The das1Δ strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480–4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the das1Δ strain on media containing methylamine or choline as a nitrogen source) suggested that DAS1 is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.     

The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1

An NADP-dependent methylene tetrahydromethanopterin (H₄MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO₂ in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H₄MPT with NADP⁺ rather than with NAD⁺, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H₄F) with NADP⁺. With methylene H₄F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (V_max/K_m) were approximately 20-fold lower than with methylene H₄MPT. Whereas the dehydrogenation of methylene H₄MPT (E₀ = −390 mV) with NADP⁺ (E₀ = −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H₄F (E₀ = −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H₄F dehydrogenases from other bacteria and eucarya and with those of methylene H₄MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).     

Acetate Production by Methanogenic Bacteria

Methanosarcina barkeri MS and 227 and Methanosarcina mazei S-6 produced acetate when grown on H₂-CO₂, methanol, or trimethylamine. Marked differences in acetate production by the two bacterial species were found, even though methane and cell yields were nearly the same. M. barkeri produced 30 to 75 μmol of acetate per mmol of CH₄ formed, but M. mazei produced only 8 to 9 μmol of acetate per mmol of CH₄.     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

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.     

Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in Methylobacterium extorquens AM1

The facultative methylotroph Methylobacterium extorquens AM1 possesses two pterin-dependent pathways for C(1) transfer between formaldehyde and formate, the tetrahydrofolate (H(4)F)-linked pathway and the tetrahydromethanopterin (H(4)MPT)-linked pathway. Both pathways are required for growth on C(1) substrates; however, mutants defective for the H(4)MPT pathway reveal a unique phenotype of being inhibited by methanol during growth on multicarbon compounds such as succinate. It has been previously proposed that this methanol-sensitive phenotype is due to the inability to effectively detoxify formaldehyde produced from methanol. Here we present a comparative physiological characterization of four mutants defective in the H(4)MPT pathway and place them into three different phenotypic classes that are concordant with the biochemical roles of the respective enzymes. We demonstrate that the analogous H(4)F pathway present in M. extorquens AM1 cannot fulfill the formaldehyde detoxification function, while a heterologously expressed pathway linked to glutathione and NAD(+) can successfully substitute for the H(4)MPT pathway. Additionally, null mutants were generated in genes previously thought to be essential, indicating that the H(4)MPT pathway is not absolutely required during growth on multicarbon compounds. These results define the role of the H(4)MPT pathway as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.     

Hydrogen Partial Pressures in a Thermophilic Acetate-Oxidizing Methanogenic Coculture

Hydrogen partial pressures were measured in a thermophilic coculture comprised of a eubacterial rod which oxidized acetate to H₂ and CO₂ and a hydrogenotrophic methanogen, Methanobacterium sp. strain THF. Zinder and Koch (S. H. Zinder and M. Koch, Arch. Microbiol. 138:263-272, 1984) originally predicted, on the basis of calculations of Gibbs free energies of reactions, that the H₂ partial pressure near the midpoint of growth of the coculture should be near 4 Pa (ca. 4 × 10⁻⁵ atm; ca. 0.024 μM dissolved H₂) for both organisms to be able to conserve energy for growth. H₂ partial pressures in the coculture were measured to be between 20 and 50 Pa (0.12 to 0.30 μM) during acetate utilization, approximately one order of magnitude higher than originally predicted. However, when ΔG_f (free energy of formation) values were corrected for 60°C by using the relationship ΔG_f = ΔH_f − TΔS (ΔH_f is the enthalpy or heat of formation, ΔS is the entropy value, and T is the temperature in kelvins), the predicted value was near 15 Pa, in closer agreement with the experimentally determined values. The coculture also oxidized ethanol to acetate, a more thermodynamically favorable reaction than oxidation of acetate to CO₂. During ethanol oxidation, the H₂ partial pressure reached values as high as 200 Pa. Acetate was not used until after the ethanol was consumed and the H₂ partial pressure decreased to 40 to 50 Pa. After acetate utilization, H₂ partial pressures fell to approximately 10 Pa and remained there, indicating a threshold for H₂ utilization by the methanogen. Axenic cultures of the acetate-oxidizing organism were combined with pure cultures of either Methanobacterium sp. strain THF or Methanobacterium thermoautotrophicum ΔH to form reconstituted acetate-oxidizing cocultures. The H₂ partial pressures measured in both of these reconstituted cocultures were similar to those measured in the original acetate-oxidizing rod coculture. Since M. thermoautotrophicum ΔH did not use formate as a substrate, formate is not necessarily involved in interspecies electron transfer in this coculture.     

The Expression of Superoxide Dismutase (SOD) and a Putative ABC Transporter Permease Is Inversely Correlated during Biofilm Formation in Listeria monocytogenes 4b G

Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.     

Utilization of Methanol plus Hydrogen by Methanosarcina barkeri for Methanogenesis and Growth

Methanosarcina barkeri grew on methanol plus H₂. Both substrates were consumed in equimolar amounts. Growth was strictly dependent on the presence of acetate, which was required for the biosynthesis of cellular constituents. Only about 0.4% of the methane produced originated from acetate. By using deuterated methanol, it was demonstrated that methanogenesis from this compound under H₂ did not occur via oxidation of methanol to CO₂ and subsequent reduction but by direct reduction with H₂. Growth yields with methanol plus H₂ and with methanol alone were not significantly different: 2.8 g of cells per mol of methanol in mineral medium and 4.6 g of cells per mol of methanol in complex medium, respectively. Growth of M. barkeri on methanol plus H₂ depended strictly on the presence of sodium ions in the medium. In the presence of 50 mM K⁺ the K_s for Na⁺ was 5 mM.     

Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed ¹⁴C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH₄ produced, but 14% of the CO₂ generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH₄. ¹⁴CH₄ was also produced from added ¹⁴CO₂, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of ¹⁴CH₄ and ¹⁴CO₂ generation from [2-¹⁴C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H₂ plus CO₂ indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH₄ from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.