Metabolic regulation in the facultative methylotroph Arthrobacter P1. Growth on primary amines as carbon and energy sources

During growth of the facultative methylotroph Arthrobacter P1 on methylamine or ethylamine both substrates are metabolized initially in an identical fashion, via the respective aldehydes. The regulatory mechanisms governing the synthesis and activities of enzymes involved in amine and aldehyde utilization were studied in substrate transition experiments. Transfer of ethylamine-grown cells into a medium with methylamine resulted in immediate exeretion of low levels of formaldehyde (max. 0.5 mM) and formate. In the reverse experiment, transfer of methylaminegrown cells into a medium with ethylamine, excretion of much higher levels of acetaldehyde (max. 3.5 mM) occurred. These different levels of aldehyde accumulation were also observed in studies with mutants of Arthrobacter P1 blocked in the synthesis of hexulose phosphate synthase or acetaldehyde dehydrogenase. In wild type Arthrobacter P1, aldehyde production resulted in rapid induction of the synthesis of enzymes involved in their degradation but also in temporary inhibition of further amine utilization and growth. The latter aetivities only resumed at normal rates after the disappearance of the aldehydes from the cultures. Acetaldehyde utilization resulted in intermittent excretion of ethanol and acetate, whereas formaldehyde utilization resulted in further accumulation of formate.During growth of Arthrobacter P1 in the presence of methylamine accumulation of toxic levels of formaldehyde is prevented because of the rapid synthesis of hexulose phosphate synthase to high activities and, in transient state situations, by feedback inhibition of formaldehyde on the activities of the methylamine transport system and amine oxidase.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoate) - HPS hexulosephosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Regulation and function of transaldolase isoenzymes involved in sugar and one-carbon metabolism in the ribulose monophosphate cycle methylotroph Arthrobacter P1

In the facultative methylotroph Arthrobacter P1 the enzyme transaldolase plays an important role in both the pentose phosphate pathway and in the ribulose monophosphate cycle of formaldehyde fixation.Among gluconate-negative mutants of Arthrobacter P1 strains occurred which also were unable to grow on xylose because they had lost the ability to synthesize transaldolase. Furthermore, this loss of transaldolase activity resulted in decreased growth rates on a number of other heterotrophic substrates. Contrary to expectation, these mutants still grew on methylamine and were even able to use gluconate as a carbon source at normal rates provided methylamine was supplied as a nitrogen source. Under these conditions high levels of transaldolase were observed.Partial purification of the transaldolases synthesized by gluconate-grown cells of wild type Arthrobacter P1 and methylamine-grown cells of one of these mutants, strain Art 98, revealed the presence of transaldolase isoenzymes. These enzymes displayed similar kinetics but were very different in heat sensitivity. Functionally these isoenzymes are apparently very similar but their synthesis is regulated differently. One of the enzymes is synthesized constitutively whereas the other is specifically induced during growth on C₁ compounds. Strain Art 98 has lost the ability to synthesize the constitutive transaldolase. It is postulated that the C₁-induced transaldolase serves to ensure a sufficiently high rate of regeneration of ribulose-5-phosphate during growth on C₁ compounds.Abbreviations RuMP ribulose monophosphate - DEAE diethylaminoethyl     

Regulation of methylotrophic and heterotrophic metabolism in Arthrobacter P1. Growth on mixtures of methylamine and acetate in batch and continuous cultures

Incubations of Arthrobacter P1 in batch culture in media with mixtures of acetate and methylamine resulted in sequential utilization of the two carbon substrates, but not in diauxic growth. Irrespective of the way cells were pregrown, acetate was the preferred substrate and subsequent studies showed that this is due to the fact that acetate is a strong inhibitor of the methylamine transport system and amine oxidase in Arthrobacter P1. An analysis of enzyme activities in cell-free extracts showed that synthesis of amine oxidase occurred already in the first growth phase with acetate, whereas rapid synthesis of hexulose phosphate synthase was only observed once methylamine utilization started. It is therefore concluded that in Arthrobacter P1 the synthesis of the enzymes specific for methylamine oxidation is not regulated co-ordinately with those involved in formaldehyde fixation, but induced sequentially by methylamine and formaldehyde, respectively.During growth of Arthrobacter P1 on the same mixture in carbon- and energy source-limited continuous cultures both substrates were used simultaneously and completely at dilution rates below the _max on either of these substrates. Addition of methylamine, in concentrations as low as 0.5 mM, to the medium reservoir of an acetate-limited continuous culture (D=0.10 h^-1) already resulted in synthesis of both amine oxidase and hexulose phosphate synthase. In the reverse experiment, addition of acetate to the medium reservoir of a methylamine-limited continuous culture (D=0.10 h^-1), acetate was initially only used as an energy source. Synthesis of the glyoxylate cycle enzymes, however, did occur at acetate concentration in the feed above 7.5–10 mM. This indicates that at acetate concentrations below 10 mM the metabolism of the C₁ substrate methylamine is able to cause a complete repression of the synthesis of the enzymes involved in carbon assimilation from the C₂ substrate acetate.Abbreviations HPS Hexulose phosphate synthase - MS mineral salts - RuMP ribulose monophosphate     

Regulation of methylamine and formaldehyde metabolism in Arthrobacter P1

The regulation of methylamine and formaldehyde metabolism in Arthrobacter P1 was investigated in carbonlimited continuous cultures. To avoid toxic effects of higher formaldehyde concentrations, formaldehyde-limited cultures were established in smooth substrate transitions from choline-limitation. Evidence was obtained that the synthesis of enzymes involved in the conversion of methylamine into formaldehyde and in formaldehyde fixation is induced sequentially in this organism. Compared to growth with methylamine the molar growth yield on formaldehyde was approximately 30% higher. This difference is mainly due to the expenditure of energy for the uptake of methylamine from the medium.The addition of a pulse of a heterotrophic substrate, glucose or acetate, to C₁ substrate-limited continuous cultures resulted in relief of carbon limitation and transient synthesis of increasing amounts of cell material. Concomitantly, a significant decrease in the specific activities of hexulose phosphate synthase was observed. However, the total activity of hexulose phosphate synthase in these cultures remained clearly in excess of that required to fix the formaldehyde that became available in time. The observed strong decrease in the specific activities of this RuMP cycle enzyme strongly suggests that its synthesis is controlled via catabolite repression exerted by the metabolism of heterotrophic substrates.Abbreviations HPS 3-Hexulose-6-phosphate synthase - HPI 3-hexulose-6-phosphate isomerase - RuMP ribulose monophosphate     

Arthrobacter P 1, a fast growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism of methylated amines

A facultative methylotrophic bacterium was isolated from enrichment cultures containing methylamine as the sole carbon source. It was tentatively identified as an Arthrobacter species. Extracts of cells grown on methylamine or ethylamine contained high levels of amine oxidase (E.C. 1.4.3.) activity. Glucose- or choline-grown cells lacked this enzyme. Oxidation of primary amines by the enzyme resulted in the formation of H₂O₂; as a consequence high levels of catalase were present in methylamine-and ethylamine-grown cells. The significance of catalase in vivo was demonstrated by addition of 20 mM aminotriazole (a catalase inhibitor) to exponentially growing cells. This completely blocked growth on methylamine whereas growth on glucose was hardly affected. Cytochemical studies showed that methylamine-dependent H₂O₂ production mainly occurred on invaginations of the cytoplasmic membrane. Assimilation of formaldehyde which is generated during methylamine oxidation was by the FBP variant of the RuMP cycle of formaldehyde fixation. The absence of NAD-dependent formaldehyde and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formal-dehyde via hexulose phosphate synthase. Enzyme profiles of the organism grown on various substrates suggested that the synthesis of amine oxidase, catalase and the enzymes of the RuMP cycle is not under coordinate control.     

Uptake of methylamine via an inducible,energy-dependent transport system in the facultative methylotroph Arthrobacter P1

Cytoplasmic membrane vesicles were prepared by a lysozyme-salt treatment from Arthrobacter P1 grown on methylamine as the carbon and energy source. In the presence of an ascorbate-phenazine methosulphate electron donor system, these vesicles accumulated methylamine in unmodified form by an inducible transport system. This system has a high affinity for methylamine (K_app=20–25 M). The effect of the ionophores valinomycin and nigericin combined with membrane potential () and pH-gradient (pH) measurements demonstrated that methylamine uptake is electrogenic and driven by the . Optimal activity is observed at pH 6.5 and 30°C. Methylamine uptake was not affected by the presence of ammonium ions but was inhibited by the primary amines ethylamine (competitively), propylamine, butylamine and benzylamine. In addition, formaldehyde and acetate, at a concentration of 1 mM, inhibited methylamine uptake almost completely. These compounds were shown to be non-competitive inhibitors. A strong inhibition observed in the presence of plumbagin could be relieved by addition of dithiothreitol. This indicates that the oxidation-reduction state of, probably, carrier dithiol-disulfide-groups is an important factor in methylamine translocation in Arthrobacter P1.     

Nitrogen metabolism in the facultative methylotroph Arthrobacter P1 grown with various amines or ammonia as nitrogen sources

The metabolism of trimethylamine (TMA) and dimethylamine (DMA) in Arthrobacter P1 involved the enzymes TMA monooxygenase and trimethylamine-N-oxide (TMA-NO) demethylase, and DMA monooxygenase, respectively. The methylamine and formaldehyde produced were further metabolized via a primary amine oxidase and the ribulose monophosphate (RuMP) cycle. The amine oxidase showed activity with various aliphatic primary amines and benzylamine. The organism was able to use methylamine, ethylamine and propylamine as carbon-and nitrogen sources for growth. Butylamine and benzylamine only functioned as nitrogen sources. Growth on glucose with ethylamine, propylamine, butylamine and benzylamine resulted in accumulation of the respective aldehydes. In case of ethylamine and propylamine this was due to repression by glucose of the synthesis of the aldehyde dehydrogenase(s) required for their further metabolism. Growth on glucose/methylamine did not result in repression of the RuMP cycle enzyme hexulose-6-phosphate synthase (HPS). High levels of this enzyme were present in the cells and as a result formaldehyde did not accumulate. Ammonia assimilation in Arthrobacter P1 involved NADP-dependent glutamate dehydrogenase (GDH), NAD-dependent alanine dehydrogenase (ADH) and glutamine synthetase (GS) as key enzymes. In batch cultures both GDH and GS displayed highest levels during growth on acetate with methylamine as the nitrogen source. A further increase in the levels of GS, but not GDH, was observed under ammonia-limited growth conditions in continuous cultures with acetate or glucose as carbon sources.Abbreviations HPS hexulose-6-phosphate synthase - RuMP ribulose monophosphate - DMA dimethylamine - TMA trimethylamine - TMA-NO trimethylamine-N-oxide - ICL isocitrate lyase - GS glutamine synthetase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOGAT glutamate synthase     

Regulation of methanol metabolism in the facultative methylotroph Nocardia sp. 239 during growth on mixed substrates in batch- and continuous cultures

The regulation of methanol metabolism in Nocardia sp. 239 was investigated. Growth on mixtures of glucose or acetate plus methanol in batch cultures resulted in simultaneous utilization of the substrates. The presence of glucose, but not of acetate, repressed synthesis of the ribulose monophosphate (RuMP) cycle enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), and methanol was used as an energy source only. Comparable results were obtained following addition of formaldehyde (fed-batch system) to a culture growing on glucose. The synthesis of the methanol dissimilatory and assimilatory enzymes in Nocardia sp. 239 thus appears to be controlled differently. Methanol and/or formaldehyde induce the synthesis of these enzymes, but under carbon-excess conditions their inducing effect on HPS and HPI synthesis is completely overruled by glucose, or metabolites derived from it. Repression of the synthesis of these RuMP cycle enzymes was of minor importance under carbon- and energy-limiting conditions in chemostat cultures. Addition of a pulse of glucose to a formaldehyde-limited (2.5 mmol l^–1 h^–1) fed-batch culture resulted in a decrease in the levels of several enzymes of methanol metabolism (including HPI), whereas the HPS levels remained relatively constant. Increasing HPS/HPI activity ratios were also observed with increasing growth rates in formaldehyde-limited chemostat cultures. The data indicate that additional mechanisms, the identity of which remains to be elucidated, are involved in controlling the levels of these C₁-specific enzymes in Nocardia sp. 239.Abbreviations HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - RuMP ribulose monophosphate - FBP fructose-1,6-bisphosphate - PFK 6-phosphofructokinase     

Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotrophArthrobacter P1

WhenArthrobacter P₁ is grown on choline, betaine, dimethylglycine or sarcosine, an NAD⁺-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa±10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa±3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the K_m values were 0.10 and 0.80 mM for formaldehyde and NAD⁺, respectively. The enzyme was heat-stable at 50° C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.Abbreviation RuMP Ribulose monophosphate     

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

Phenylalanine and tyrosine metabolism in the facultative methylotroph Nocardia sp. 239

Nocardia sp. 239 is able to use l-tyrosine and both d- and l-phenylalanine as carbon-, energy- and nitrogen sources for growth. The catabolism of these compounds is by way of (4-hydroxy)phenylpyruvate and (4-hydroxy)-phenylacetate as intermediates and the pathways merge at the level of homogentisate. The conversion of the amino acids into (4-hydroxy)phenylpyruvate is catalyzed by an inducible NAD-dependent phenylalanine dehydrogenase and l-tyrosine aminotransferase, respectively. Incubation of the organism in media with l-phenylalanine plus phenyl-pyruvate resulted in diauxic growth, with phenylpyruvate used first. Phenylalanine dehydrogenase activity cold only be detected after depletion of phenylpyruvate, in the ensuing second growth phase on l-phenylalanine. During growth on phenylalanine plus methanol, low levels of phenylalanine dehydrogenase were detected and this resulted in simultaneous utilization of the two substrates. Following diepoxyoctane treatment, mutants of Nocardia sp. 239 affected in phenylalanine and phenylpyruvate degradation were isolated. Double mutants blocked in both phenylalanine dehydrogenase and phenylpyruvate decarboxylase completely failed to catabolize phenylalanine. The absence of these enzymes did not affect growth on tyrosine.Abbreviations RuMP ribulose monophosphate - EMS ethylmethanesulphonate - NTG N-methyl-N-nitro-N-nitrosoguanidine     

Purification,characterization and regulation of a monomeric l-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min^-1 mg^-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPT hexulose-6-phosphate isomerase - FPLC fast protein liquid chromatography     

Isolation and characterization of a fructosyl-amine oxidase from an <Emphasis Type="Italic">Arthrobacter </Emphasis>sp.

An Arthrobacter sp. was isolated that, when induced by fructosyl-valine, expressed a fructosyl-amine oxidase (FAOD) that was specific for -glycated amino acids. The N-terminal amino acid sequence of the purified oxidase was determined and used to design oligonucleotides to amplify the gene by inverse PCR. Expression of the gene in Escherichia coli produced 0.23 units FAOD per mg protein, over 30-fold greater than native expression levels, with properties almost indistinguishable from the native enzyme. The presence of FAOD was confirmed in other Arthrobacter ssp.Revisions requested 8 September 2004; Revisions received 4 November 2004     

Isolation and characterization of Streptomyces lividans mutants deficient in intraplasmid recombination

Summary Plasmid pIF132 containing two direct repeats of the mel (melanin) sequence was used to monitor intraplasmid recombination. Five mutants of Streptomyces lividans TK64 deficient in intraplasmid recombination were isolated. Four contained additional defects in aerial mycelium formation, pigmentation, and nutrient requirements; among these two showed extensive amplification of chromosomal sequences. Mutant JT46 had no pleiotropic defects but had the most severe blockage in recombination. Only one of the mutants was slightly more sensitive to UV and two were slightly more sensitive to mitomycin C. Plasmid pWCL1 (containing pIJ702, pUC12, and HBVsAg sequences; Lee et al. 1986) could not stably replicate in TK64 without spontaneous deletions. In contrast the mutant JT46 maintained the integrity of pWCL1 much more stably.     

Mapping of some genes involved in C-1 metabolism in the facultative methylotroph Methylobacterium sp strain AM1 (Pseudomonas AM1)

R68.45 mediated mobilisation of the chromosome of Methylobacterium sp strain AM1 has been investigated. High frequencies of cotransfer of four genes required for C-1 metabolism with the genes coding for streptomycin, phosphonomycin and cycloserine resistance were demonstrated. A preliminary map of this region has been constructed on the basis of the results of three and four factor crosses showing that not all the C-1 genes are contiguous.Abbreviations Str streptomycin - Pho phosphonomycin - Cyc cycloserine - Tc tetracycline - Km kanamycin - Cb carbenicillin - Ade adenine - Thi thiamine - Met methionine     

Intramolecular electron transfer in the oxidation of amines by methylamine oxidase from Arthrobacter P1

 The intramolecular electron-transfer rate constant for the Cu(II)–topa_NH2⇌ Cu(I)–topa_SQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated k_obs = 150±30 s^–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond to a forward electron-transfer rate constant k_ET≈ 60–75 s^–1. Although substantially slower than the previously determined k_ET≈ 20 000 s^–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that k_ET > k_cat≈ 4–10 s^–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover. Received: 16 August 1995 / Accepted: 21 December 1995     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Occurrence of cytochrome c-554 in a facultative methylotroph,Protaminobacter ruber strain NR-1, during bacteriochlorophyll formation

A facultative methylotroph, Protaminobacter ruber was grown under two different conditions (aerobically grown under light, and aerobically in the dark after a light period). Bacteriochlorophyll was synthesized inducibly in the cells which were initially grown in the ligt and then grown in the dark, while bacteriochlorophyll was not found in the cells cultured under continuous light. Cytochrome c-554 was solely synthesized parallel to bacteriochlorophyll after switching from light to dark conditions. Both cytochrome c-554 and bacteriochlorophyll levels in the membrane preparation reached to a plateau in 24 h after switching from light and dark conditions. This cytochrome was membrane-bound and its M _r was 45,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis. The midpoint potential was 358 mV at pH 7. Other major membrane-bound cytochromes and two soluble cytochromes were present in both types of cells and their content did not change irrespective of growth conditions.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Bchl bacteriochlorophyll     

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