The role of blue copper proteins in the oxidation of methylamine by an obligate methylotroph.

Organism 4025, an obligate methylotroph, when grown on methylamine in the presence of a high concentration of copper, contained high concentrations of methylamine dehydrogenase and two blue copper proteins, amicyanin and an azurin-type protein; these were purified to homogeneity and characterized. The methylamine dehydrogenase is a basic protein (pI 8.8) and consists of light and heavy subunits (Mr 14100 and 43000; total Mr 112000). This dehydrogenase differed slightly from other methylamine dehydrogenases in its absorption spectrum and in its lack of thermal stability. Amicyanin, the more abundant blue copper protein, had an Mr of 11500, a midpoint redox potential of 294mV at pH 7.0, and a much lower isoelectric point (pI5.3) than other amicyanins. Its absorption maximum was 620 nm (7-24 nm higher than those of other amicyanins); its absorption coefficient (at 620 nm) was 3.8 mM-1 X cm-1. The 'azurin' (6% of the blue copper protein) had an Mr of 12500, a midpoint redox potential of 323 mV and a high isoelectric point (pI 9.4). Its absorption maximum was 620 nm, the absorption coefficient (16 mM-1 X cm-1) at this wavelength being considerably greater than that of any blue copper protein described previously. The partially-purified soluble cytochromes cH and cL were similar to those of other methylotrophs. The interactions of the purified redox proteins were investigated in order to elucidate their role in methylamine oxidation. Methylamine dehydrogenase was able to donate electrons only to amicyanin, the rate of reaction being 2.04 mmol/min per mumol of methylamine dehydrogenase; this is sufficient to account for the rate of respiration in whole bacteria. The blue copper proteins were able to react rapidly with each other and with both the soluble cytochromes c.     

The electron-transport chains of the obligate methylotroph Methylophilus methylotrophus

The cytochrome complement of Methylophilus methylotrophus and its respiratory properties were determined during batch culture and in continuous culture under conditions of methanol-, nitrogen- and O₂-limitation. About 35% of the cytochrome c produced by the bacteria was released into the growth medium, and of the remaining cytochrome c about half was membrane-bound and half was soluble. Two cytochromes c were always present on membranes (redox potentials 375mV and 310mV), and these probably correspond to the soluble cytochromes c described previously [Cross & Anthony (1980) Biochem. J. 192, 421–427]. A third minor component of cytochrome c (midpoint potential 356mV) was only detected on membranes of methanol-limited bacteria. M. methylotrophus always contained two membrane-bound cytochromes b with α-band absorption maxima of about 556 and 563nm (measured at room temperature) and midpoint potentials of 110 and 60mV respectively. There appeared to be relatively more of the cytochrome b₅₆₃ in methanol-limited bacteria. A third b-type cytochrome with an α-band absorption maximum at 558 (at 77K) reacted with CO and had a high midpoint redox potential (260mV); it is thus a potential oxidase and hence is called cytochrome o. The roles of these cytochromes in electron transport were confirmed by investigating the patterns of respiratory inhibition. It is proposed that two cytochromes are physiological oxidases: cytochrome a+a₃, which is present only in methanol-limited conditions, and the cytochrome o, which is induced 10-fold in conditions of methanol excess. Schemes for electron transport from methanol and NAD(P)H to O₂ in M. methylotrophus under various limitations are proposed. Spectra and potentiometric titrations of cytochromes in whole cells and membranes of M. methylotrophus grown under various nutrient limitations have been deposited as Supplementary Publication SUP 50111 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Methylamine dehydrogenase from the obligate methylotroph Methylomonas methylovora.

An obligate methyltroph Methylomonas methylovora oxidized methylamine, formaldehyde, and formate. Enzymes oxidizing these substrates were detected in a cell-free system. Phenazine methosulfate-linked methylamine dehydrogenase was purified 21-fold. The enzyme had optimum activity at pH 7.5 and was stable at 60 degrees C for 5 min. The enzyme activity was inhibited by parachloromercuric benzoate, isonicotinic acid hydrazide, mercuric chloride, and sodium borate.     

Proteolysis in the obligate methylotroph Methylophilus methylotrophus

This report represents the first demonstration of degradation of intracellular protein in the obligate methylotroph, Methylophilus methylotrophus. Proteolysis in batch culture was followed by a pulse-chase protocol which included chloramphenicol during the chase period to prevent re-incorporation of the radio-label, l-[4,5-³H] isoleucine. Starvation for a nitrogen source mildly stimulated proteolysis whereas starvation for the carbon source (0.5% v/v methanol) inhibited proteolysis by over 50%. Respiratory inhibitors (e.g. 2,4-DNP) caused a rapid decline in both intracellular ATP concentration and protein catabolism. Proteins synthesized after the addition of methanol (5% v/v) and ethanol (5% v/v) to the growth medium were subject to rapid degradation. Breakdown of abnormal proteins generated by treatment with dihydrostreptomycin and puromycin was also inhibited by inhibitors of respiration and deprivation of carbon source. The stability of an heterologous gene product, interferon -2, was also investigated; loss of immunoreactivity was reduced in the absence of methanol but not prevented.     

Respiration-linked proton translocation in the obligate methylotroph Methylophilus methylotrophus.

The stoicheiometries of respiration-linked proton translocation in Methylophilus methylotrophus were determined by using both the oxygen-pulse and initial-rate methods. The latter has also been used to determine leads to charge/O quotients (measured as yield K+/O quotients) in order to ascertain whether the leads to H+/O quotients might be underestimated by H+/anion symport. The results suggest that 6H+/O are translocated during NADH oxidation, and that 2H+/O are translocated during the oxidation of methanol to formaldehyde. There was no evidence for underestimation of the leads to H+/O quotients due to H+/anion symport, except by the movement of formic acid during formate oxidation. By comparing these results with the known growth efficiencies of this organism, an leads to H+/ATP quotient of close to 2 g-ions of H+/mol of ATP can be calculated. It is proposed that the respiratory chain in Methylophilus methylotrophus is arranged such that there are three sites of energy conservation for NADH oxidation, each translocating 2H+ and each linked to the synthesis of one molecule of ATP. Only the third site of energy conservation is involved in methanol oxidation.     

Respiration of an obligate methylotroph in the presence of various substrates]

Respiration of the cells of Methylococcus ucrainicus, strain 21, cultivated in the atmosphere of methane, is stimulated by methanol, formaldehyde, formate, n-alcohols, and allyl alcohol. The rate of oxygen assimilation is lower in the presence of isopropanol, isobutanol, propane, butane, maltose, and some organic acids (acetate, fumarate, citrate, succinate). The Michaelis constant for methanol is 88 mcM. Oxidation of methane, methanol, formaldehyde, and formate by the bacterium is inhibited by cyanide, hydroxylamine, and azide. The rate of oxygen assimilation by the cells in the presence of methane and other C1-compounds did not decrease after the suspension had been stored at 4 degrees C during four months and longer.     

Citrate synthase from the obligate methylotroph Methylobacillus flagellatum

Abstract Formation of the lesion in the Escherichia coli inner membrane caused by λ lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the λ R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.     

Characterisation of the electron transport chain of an obligate methylotroph,strain 4025

• 1.1. The obligate methanol-utilising bacterium strain 4025 contains cytochromes b and c. Cytochrome a is never present.
• 2.2. The soluble cytochrome c is similar to that from other methylotrophs in reacting (slowly) with carbon monoxide and it can be separated into two types, differing markedly in their isoelectric points.
• 3.3. Some of the cytochrome b reacts rapidly with carbon monoxide and is thus the likely cytochrome oxidase (cytochrome o).
• 4.4. The partially purified, NAD⁺-independent methanol dehydrogenase is similar to such enzymes from the other methanol-utilising bacteria in respect of its prosthetic group, dependence on ammonia or methylamine for activity and its wide substrate specificity.
• 5.5. The fluorescence seen in colonies of this organism is probably due to a flavin derivative.
• 6.6. This study of electron transport components does not shed any light on the unusually high copper requirement shown by this methylotroph.

     

One-carbon compounds transport studies in the obligate methylotroph

Cells of obligate methylotrophic Gram-negative bacterium Methylobacillus flagellatum KT which can only grow on methanol and methylamine media possess three different carriers mediating uptake of methylamin depending on growth conditions. All three uptake systems are energy-dependent, the methylamine uptake was inhibited by oxidative phosphorylation uncoupler and respiratory inhibitors. The first active transport system for methanol in the cells of obligate methylotroph was also demonstrated. The parameters of this system were measured, their dependence on energy, presence of respiratory inhibitors and uncoupler was shown.Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexyl-carbodiimide     

Refined genetic map of the obligate methylotroph Methylobacillus flagellatum

We present a refined genetic map of the obligate methylotroph Methylobacillus flagellatum. New, Hfr (high-frequency-of-transfer) donors, and pulsed-field gel electrophoresis, were used to determine that M.?flagellatum contains one ～3.1-Mb circular chromosome, and no plasmids. A correlation between time-of-entry units and DNA length was established. Using in vivo and in vitro cloning, and sequencing, a number of new genetic markers were identified and mapped; in addition, the nature of some of the previously mapped markers was elucidated.     

l-threonine transport in the obligate methylotroph Methylobacillus flagellatum KT

Abstract The accumulation of l -threonine by the methylotrophic bacterium Methylobacillus flagellatum KT occurs via a specific system that is capable of transporting l -threonine against a 100-fold concentration gradient. This transport system demonstrates the following kinetic parameters: K _m= 0.2 mM and V _max= 2.5 nmol/min/mg of cells (dry weight). The activity of the system is inhibited by oxidative phosphorylation uncouplers and valinomycin. Cytoplasmic l -threonine does not leak from the cell, but bacteria are capable of exchanging exogenous l -threonine for its intracellular counterpart.     

Regulation of phenylalanine biosynthesis in the obligate methylotroph Methylobacillus M75

Regulation of phenylalanine biosynthesis has been studied in the bacterium Methylobacillus M75 on the level of enzymes 3-deoxy-D-arabinoheptulose-7-phosphate-synthase, chorismatmutase, prephenatdehyrataze. The DAHP-synthase is shown to be synthesized constitutively and its activity is inhibited by all aromatic aminoacids and antranilate. The synthesis of chorismatmutase and prephenatdehydratase is repressed by tyrosine, the activity of the latter enzyme, besides that, is inhibited by phenylalanine, the effect of which is decreased in the presence of tyrosine.     

Refined genetic map of the obligate methylotroph Methylobacillus flagellatum

We present a refined genetic map of the obligate methylotroph Methylobacillus flagellatum. New, Hfr (high-frequency-of-transfer) donors, and pulsed-field gel electrophoresis, were used to determine that M.␣flagellatum contains one ∼3.1-Mb circular chromosome, and no plasmids. A correlation between time-of-entry units and DNA length was established. Using in vivo and in vitro cloning, and sequencing, a number of new genetic markers were identified and mapped; in addition, the nature of some of the previously mapped markers was elucidated. Received: 11 August 1997 / Accepted: 11 December 1997     

Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions

The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes. As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases. The NADP⁺-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol l^-1 of ammonium in the growth medium) and increased in response to an increase in nitrogen availability. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation. In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h^-1, and then sharply dropped. In the N-sufficient cultures both NAD⁺- and NADP⁺-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated. Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities. When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased. When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3-hours delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse.     

The cytochrome cbo from the obligate methylotroph Methylobacillus flagellatus KT is a cytochrome-c oxidase

The oxidase cho of Methylobacillus flagellatus KT was purified to homogeneity by nondenaturing gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cbo with a pH optimum of 8.3. When TMPD served as electron donor for the oxidase cho, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and of only ascorbate. The kinetic constants, determined at pH 7.0, were as follows: oxidation by the enzyme of reduced TMPD at pH 7.0 was characterized by KM = 0.86 mM and Vmax = 1.1 mumol O2/(min mg protein), and oxidation of reduced cytochrome c from horse heart was characterized by KM = 0.09 mM and Vmax = 0.9 mumol O2/(min mg protein) Cyanide inhibited ascorbate/TMPD oxidase activity (Ki = 4.5-5.0 microM). The soluble cytochrome cH (12 kDa) partially purified from M. flagellatus KT was found to serve as the natural electron donor for the oxidase cbo.     

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.     

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus

The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.     

Purification and characterization of azurin from the methylamine-utilizing obligate methylotroph Methylobacillus flagellatus KT

Methylamine dehydrogenase (MADH) and azurin were purified from the periplasmic fraction of the methylamine-grown obligate methylotroph Methylobacillus flagellatus KT. The molecular mass of the purified azurin was 16.3 kDa, as measured by SDS-PAGE, or 13 920 Da as determined by MALDI-TOF mass spectrometry. Azurin of M. flagellatus KT contained 1 copper atom per molecule and had an absorption maximum at 620 nm in the oxidized state. The redox potential of azurin measured at pH 7.0 by square-wave voltammetry was +275 mV versus normal hydrogen electrode. MADH reduced azurin in the presence of methylamine, indicating that this cupredoxin is likely to be the physiological electron acceptor for MADH in the electron transport chain of the methylotroph. A scheme of electron transport functioning in M. flagellatus KТ during methylamine oxidation is proposed.     

(14C)acetate assimilation by a type I obligate methylotroph, Methylococcus capsulatus.

Methanol and formate oxidation supported the assimilation of [14C]acetate by cell suspensions of Methylococcus capsulatus; oxidation of other primary alcohols, except ethanol, did not. The extent of [1-14C]acetate assimilation supported by methanol oxidation was decreased in the presence of primary alcohols, except ethanol. Potassium cyanide (0.33 mM) completely inhibited the oxidation of formate and its stimulation of [1-14C]acetate assimilation. The amount of [1-14C]acetate assimilation supported by methanol oxidation was significantly inhibited by cyanide.