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At least 19 recordsLinked to original sources
PubMed1975

Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs.

The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.

Bacteria↗
PubMed1975

Regulation of citrate synthase activity in methylotrophs by reduced nicotinamide-adenine dinucleotide, adenine nucleotides and 2-oxoglutarate.

Citrate synthase from two typical facultative methylotrophs, but not from four obligate methylotrophs or from two restricted facultative methylotrophs, is inhibited by 0.1 mM-NADH. ATP or ADP (both at 10mM) inhibits all the citrate synthases, whereas 2-oxoglutarate (10mM) inhibits those from only three of the eight methylotrophs.

Adenine Nucleotides↗
PubMedDate not supplied

[Pyruvate and phosphoenolpyruvate carboxylase in methylotrophs].

The activity of pyruvate and phosphoenolpyruvate carboxylases was determined in cell extracts of obligate and facultative methylotrophs which metabolized monocarbon reduced compounds via different pathways. Phosphoenolpyruvate carboxylase was found to be the only enzyme responsible for the high level of CO2 fixation by methylotrophs with the serine pathway (Methylosinus trichosporium, Hyphomicrobium vulgare, Pseudomonas methylica). Methylotrophs with the hexulose phosphate pathway Mehylobacter chroococcum, Methylomonas methanica, Pseudomonas oleovorans, Arthrobacter globiformis) and yeast (Candida methylica) assimilated less CO2 but contained more enzymes involved in arboxylation of phosphoenolpyruvate (phosphoenolpyruvate carboxylase, EG 4.1.1.31; phosphoenolpyruvate carboxykinase, EC 4.1.1.32) or pyruvate (pyruvate carboxylase, EC 6.4.1.1; malic-enzyme, EC 4.1.1.40). Phosphoenolpyruvate carboxytransphosphorylase (EC 4.1.1.38) was not found in any of the studied strains. The properties and the role of carboxylases in the metabolism of methylotrophs are discussed.

Arthrobacter↗
PubMed1979

Tricarboxylic acid-cycle enzymes and ATP pool in facultative and obligate methylotrophs: Pseudomonas J26 and Methylomonas Pl1.

1. No essential differences were found in the activities of tricarboxylic acid-cycle enzymes in the newly isolated facultative methylotroph Pseudomonas J26 and obligate methylotroph Methylomonas Pl1. 2-Oxoglutarate dehydrogenase and succinate dehydrogenase were absent in Methylomonas Pl1; in Pseudomonas J26 the functioning of the cycle was imparied only on the methanol medium. Citrate synthase of both organisms showed low sensitivity to 2-oxoglutarate, NADH and ATP. 2. In both methylotrophs, methanol dehydrogenase was inhibited non-competitively by ATP: the activity was reduced by half by ATP at a concentration of 5 mM. 3. Concentration of ATP in the log-phase cultures of Methylomonas Pl1 was about twice as high as in Pseudomonas J26 (4.7 and 1.7 mumol/g dry wt., respectively). 4. Differences between the energy state of Methylomonas Pl1 and Pseudomonas J26 might be due to the higher ability of the former to oxidize methanol and/or lower energy requirement for C1 assimilation by the hexulose pathway in the obligate methylotroph.

Adenosine Triphosphate↗
PubMedDate not supplied

[Ability of obligate methylotrophs to perform nitrogen fixation].

The ability for nitrification was studied among mesophilic and thermophilic cultures of obligate methylotrophs Methylobacter ucrainicus, Methylomonas methanica, and Methylococcus thermophilus. The strains were almost incapable of nitrification under autotrophic conditions. In the presence of methane, however, they oxidized NH+4 TO NO-2: over 150 mg/litre NO-2 nitrogen was found in the cultural broth. Therefore, obligate methylotrophs are capable of heterotrophic nitrification. The level of nitrification suggests that it is not a side reaction but a necessary stage of the metabolism of obligate methylotrophs. The phenotype relation between obligate methylotrophs and nitrifying organisms is discussed.

Bacteria↗
PubMedDate not supplied

[Nomenclature of obligate methylotrophs].

The nomenclature of obligate methylotrophs, i. e. bacteria using only reduced monocarbon compounds (methane, methanol, methylamines) as a carbon source, is dicussed. The chronology of naming taxons of methane oxidizing bacteria is presented and the rightfulness of their names is analyzed according to the rules of the International Codex of Bacterial Nomenclature. Such names as Methylomonas and others which are employed while describing various physiological groups of bacteria are used in the nomenclature of obligate methylotrophs, and this may be the source of errors. Therefore, only the genus of methane oxidizing bacteria should be referred to as Methylomonas whereas obligate methanol and methylamine assimilating bacteria should be classed as individual genera. The described species of methane oxidizing bacteria can be included into the genera Methylomonas, Methylococcus, Methylosinus, and Methylocystis. One should avoid such names as Methanomonas margaritae, Methanomonas immobilis, Pseudomonas methanica etc. since they do not possess the nomenclature status. All bacteria that depend obligatorily on the presence of reduced C1-compounds should be included into the family Methylomonadaceae. The taxonomy and nomenclature of facultative methylotrophs must be based on principles accepted for other organotrophic microorganisms as it has been suggested at the II International Symposium on the Growth of Microorganisms on C1-compounds (Pushchino, USSR, 1977).

Methylococcaceae↗
PubMed1979

(1-14C) acetate assimilation by obligate methylotrophs, Pseudomonas methanica and Methylosinus trichosporium.

The oxidation of one carbon compounds (methane, methanol, formaldehyde, formate) and primary alcohols (ethanol, propanol, butanol) supported the assimilation of [1-14C]acetate by cell suspensions of type I obligate methylotroph, Pseudomonas methanica, Texas strain, and type II obligate methylotroph, Methylosinus trichosporium, strain PG. The amount of oxygen consumed and substrate oxidized correlated with the amount of [1-14C]acetate assimilated during oxidation of C-1 compounds and primary alcohols. Oxidation of methanol, formaldehyde, and primary alcohols in extracts of Pseudomonas methanica, Texas strain, and Methylosinus trichosporium, strain PG, was catalyzed by a phenazine methosulfate linked, ammonium ion dependent methanol dehydrogenase. The oxidation of aldehydes was catalyzed by a phenazine methosulfate linked, ammonium ion independent aldehyde dehydrogenase. Formate was oxidized by a NAD+ linked formate dehydrogenase.

Acetates↗
PubMed2025

Lanthanide-dependent isolation of phyllosphere methylotrophs selects for a phylogenetically conserved but metabolically diverse community.

Lanthanides have emerged as important metal cofactors for biological processes. Lanthanide-associated metabolisms are well-studied in leaf symbiont methylotrophic bacteria, which utilize reduced one-carbon compounds such as methanol for growth. Yet, the importance of lanthanides in plant-microbe interactions and on microbial physiology and colonization in plants remains poorly understood. To investigate this, 344 pink-pigmented facultative methylotrophs were isolated from soybean leaves by selecting for bacteria capable of methanol oxidation with lanthanide cofactors, but none were obligately lanthanide-dependent. Phylogenetic analyses revealed that all strains were nearly identical to each other and are part of the extorquens clade of Methylobacterium, despite variability in genome and plasmid sizes. Strain-specific identification was enabled by the higher resolution provided with rpoB compared to 16S rRNA as marker genes. Despite the low strain-level diversity, the metabolic capabilities of the collection diverged greatly. Strains encoding identical lanthanide-dependent alcohol dehydrogenases displayed significantly different growth rates and/or final ODs from each other on alcohols in the presence and absence of lanthanides. Several strains also lacked well-characterized lanthanide-associated genes thought to be important for phyllosphere colonization. Additionally, 3% of our isolates were capable of growth on sugars and 23% were capable of growth on aromatic acids, substantially expanding the range of substrates utilized by Methylobacterium extorquens in the phyllosphere. Our findings suggest that the expansion of metabolic capabilities, as well as differential usage of lanthanides and their influence on metabolism, among closely related strains point to evolution of niche partitioning strategies to promote colonization of the phyllosphere.

Journal Article↗
PubMed1978

Fluorescent pigments in the newly isolated methylotrophs: Pseudomonas J16 and Methylomonas Pl1.

The pigments showing fluorescence maxima at 390, 366, 450-460 and 520 nm at excitation wavelength 254, 366 and 450 nm respectively, were detected in the cells and culture media of the obligate methylotroph Methylomonas Pl1 and facultative methylotroph Pseudomonas J26. The maximum at 520 nm is associated with the occurrence of a flavin pigment enabling growth of Lactobacillus casei E ATCC-7469 on the vitamin B2 deficient medium. The remaining fluorescence maxima are related to the prosthetic group of methanol dehydrogenase.

Alcohol Oxidoreductases↗
PubMed1979

Alternative carboxylation reactions in type II methylotrophs and the localization of carboxylase activities in the intra-cytoplasmic membranes.

The peripheral arrangements of the intra-cytoplasmic membranes (ICM) as well as the guanine + cytosine (G + C) content of 63% indicate that our obligate methanotrophic strain M 102 would belong to the type II Methylotrophs. There are no active transport mechanisms for some organic additives, some of which are known to stimulate CO2-fixation. The CO2-acceptor molecules, Pyr (pyruvate) and PEP (phospho-enolpyruvate), must be metabolically intracellularly furnished. More than one carboxylating system could be induced in cell fractions. The carboxylating activities were variably registered in cell-free extracts and fragments. Indications that the site of the carboxylating activities are the ICM are discussed. The fact that the CO2-fixation intensities decreased at about the middle of the active growing phase indicates that the formation of the intermediate C4-compound OAA (oxaloacetate) must be furnished through some reactions by-passing the PEP-carboxylation step. The common serine-pathway for C1-assimilation is rearranged so as to fit in the discussed findings.

Carbon Dioxide↗
PubMed1979

Effect of growth conditions on the involvement of cytochrome c in electron transport, proton translocation and ATP synthesis in the facultative methylotroph Pseudomonas AM1.

The stoicheiometry of proton translocation, the amounts of cytochromes firmly bound to membranes, and cell yields with respect to succinate and O(2) have been measured in the facultative methylotroph Pseudomonas AM1 and in the mutant lacking cytochrome c (mutant PCT76) during carbon-limited growth and carbon-excess growth. -->H(+)/O ratios during endogenous respiration of about 4 were measured in wild-type bacteria grown in carbon-excess conditions, and in the mutant in all growth conditions. During methanol- or succinate-limited growth of wild-type bacteria the -->H(+)/O ratio increased to about 6. Cell yields with respect to succinate and O(2) were higher in wild-type than in the mutant lacking cytochrome c by an amount suggesting loss in the mutant of 30% of the ATP-generating capacity of wild-type bacteria. During carbon-limited growth on methanol or succinate some cytochrome c was tightly bound to bacterial membranes, whereas none was tightly bound in bacteria grown in batch-culture or in NH(4) (+)-limited conditions. It is proposed that the role of cytochrome c in Pseudomonas AM1 depends on growth conditions and hence on the ;needs' of the bacteria. During growth in carbon-excess conditions it is only required for methanol oxidation, mediating between methanol dehydrogenase and cytochrome a/a(3). In these conditions oxidation of NADH and succinate by way of cytochrome b and cytochrome a/a(3) occurs without the mediation of cytochrome c. This is the only route for oxidation of NADH and succinate in the cytochrome c-deficient mutant in all growth conditions. During carbon-limited growth the cytochrome c becomes bound to the membrane in such a way that it can mediate between cytochromes b and a/a(3), hence becoming involved in proton translocation and ATP synthesis during NADH and succinate oxidation. An alternative possibility is that in wild-type bacteria the cytochrome c is always involved in electron transport, but that its involvement in measurable proton translocation only occurs in carbon-limited conditions.

Adenosine Triphosphate↗
PubMed1979

NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.

Aldehyde Oxidoreductases↗
PubMed1978

Formaldehyde incorporation by a new methylotroph (L3).

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.

Alcohol Oxidoreductases↗
PubMed1979

Microbial oxidation of gaseous hydrocarbons: epoxidation of C2 to C4 n-alkenes by methylotrophic bacteria.

Over 20 new cultures of methane-utilizing microbes, including obligate (types I and III) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL M1 NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation.

Alkenes↗
PubMed1975

Serine transhydroxymethylase isoenzymes from a facultative methylotroph.

Two serine transhydroxymethylase activities have been purified from a facultative methylotrophic bacterium. One enzyme predominates when the organism is grown on methane or methanol as the sole carbon and energy source, whereas the second enzyme is the major isoenzyme found when succinate is used as the sole carbon and energy source. The enzyme from methanol-grown cells is activated by glyoxylate, is not stimulated by Mg2+, Mn2+, or Zn2+, and has four subunits of 50,000 molecular weight each. The enzyme from succinate-grown cells is not activated by glyoxylate and is stimulated by Mg2+, Mn2+, and Zn2+, and sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that this enzyme has subunit molecular weight of 100,000, the same as the molecular weight obtained for the active enzyme. Cells grown in the presence of both methanol and succinate incorporate less methanol carbon per unit time than cells grown on methanol and have a lower specific activity of the glyoxylate-activated enzyme than methanol-grown cells. Adenine, glyoxylate, or trimethoprim in the growth medium causes an increased level of serine transhydroxymethylase in both methanol- and succinate-grown cells by stimulating the synthesis of the glyoxylate-activated enzyme.

Adenine↗
PubMed1978

Alcohol dehydrogenases from a facultative methylotrophic bacterium.

Alcohol-oxidizing enzymes of the facultative methylotroph PAR were investigated after growth of the bacteria on methanol and ethanol. During methanol growth only a phenazine methosulfate-linked alcohol dehydrogenase was detected. This enzyme had broad specificity for primary alcohols and was also capable of oxidation of secondary alcohols. It had a molecular weight of 112,000, was composed of two subunits of equal molecular weight, and showed an absolute requirement for ammonium ion for activation. During ethanol growth this enzyme was absent and was replaced by a typical nicotinamide adenine dinucleotide-linked alcohol dehydrogenase of molecular weight 150,000. The latter enzyme also had broad specificity but could not oxidize methanol. This enzyme was not found during methanol growth. These data show that the organism has two distinctly separate mechanisms for oxidation of alcohols.

Alcohol Oxidoreductases↗
PubMed1979

Inhibition by itaconate of growth of methylotrophic bacteria.

The effects of the isocitrate lyase-directed growth inhibitor itaconate on the growth of certain methylotrophic organisms was investigated. It was found that growth of those organisms possessing the Icl(+)-serine pathway of one-carbon metabolism was inhibited during growth on methylamine and on acetate, but not on glucose. Organisms possessing the Icl(-)-serine pathway pathway were unaffected. Organism PAR, an Icl(-)-serine pathway type, was not specifically inhibited during growth on acetate. This finding further substantiates previous reports of the lack of isocitrate lyase in this organism, indicating a totally new pathway for acetate assimilation.

Acetates↗