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structural aspects of the dye-linked alcohol dehydrogenase of rhodopseudomonas acidophila.1. a dye-linked alcohol dehydrogenase was purified 60-fold from extracts of rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. the properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. the enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. the ami ...1979229820
characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion.methanol dehydrogenase (mdh) from methylobacterium extorquens, methylophilus methylotrophus, paracoccus denitrificans and hyphomicrobium x all contained a single atom of ca2+ per alpha 2 beta 2 tetramer. the role of ca2+ was investigated using the mdh from methylobacterium extorquens. this was shown to be similar to the mdh from hyphomicrobium x in having 2 mol of prosthetic group (pyrroloquinoline quinine; pqq) per mol of tetramer, the pqq being predominantly in the semiquinone form. mdh isolat ...19921332681
16s ribosomal rna sequence analysis for determination of phylogenetic relationship among methylotrophs.16s ribosomal rnas (rrna) of 12 methylotrophic bacteria have been almost completely sequenced to establish their phylogenetic relationships. methylotrophs that are physiologically related are phylogenetically diverse and are scattered among the purple eubacteria (class proteobacteria). group i methylotrophs can be classified in the beta- and the gamma-subdivisions and group ii methylotrophs in the alpha-subdivision of the purple eubacteria, respectively. pink-pigmented facultative and non-pigmen ...19901693657
electron-transfer flavoproteins from methylophilus methylotrophus and bacterium w3a1. 19902126331
[analysis of dna-dna homologies in obligate methylotrophic bacteria].the genotypic affinity of 19 bacterial strains obligately dependent on methanol or methylamine as carbon and energy sources was studied by techniques of molecular dna hybridization. the high homology level (35-88%) between motile strain methylophilus methanolovorus v-1447d and nonmotile strain methylobacillus sp. vsb-792 as well as other motile strains (pseudomonas methanolica atcc 21704, methylomonas methanolica nrrl 5458, pseudomonas sp. w6, strain a3) indicates that all of them belong to one ...19882463459
studies on electron transfer from methanol dehydrogenase to cytochrome cl, both purified from hyphomicrobium x.ferricytochrome cl isolated from hyphomicrobium x is an electron acceptor in assays for homologous methanol dehydrogenase (mdh), albeit a poor one compared with artificial dyes. the intermediates of mdh seen during the reaction are identical with those observed with wurster's blue as electron acceptor, indicating that the reaction cycles are similar. the assay showed a ph optimum of approx. 7.0 and scarcely any stimulation by nh4cl, this being in contrast with assays with artificial dyes, where ...19892537627
preliminary x-ray crystallographic study of methanol dehydrogenase from methylophilus methylotrophus.single crystals of methanol dehydrogenase from methylophilus methylotrophus have been prepared by the macroseeding method. the crystals belong to the monoclinic space group c2, and have unit cell parameters a = 125.62 a, b = 63.83 a, c = 83.99 a, and beta = 93.24 degrees. there is one 62,000 mr monomer in the asymmetric unit. the crystals diffract to beyond 2.0 a resolution.19863098983
electron transfer flavoprotein from methylophilus methylotrophus: properties, comparison with other electron transfer flavoproteins, and regulation of expression by carbon source.when grown on methylated amines as a carbon source, methylophilus methylotrophus synthesizes an electron transfer flavoprotein (etf) which is the natural electron acceptor of trimethylamine dehydrogenase. it is composed of two dissimilar subunits of 38,000 and 42,000 daltons and 1 mol of flavin adenine dinucleotide. it was reduced by trimethylamine dehydrogenase to a stable anionic semiquinone form, which could not be converted, either enzymatically or chemically, to the fully reduced dihydroqui ...19863711024
expression of eukaryotic coding sequences in methylophilus methylotrophus. 19826803176
[properties of the obligate methylotroph methylophilus methanolovorus].a strain of obligate methylotrophic bacteria utilizing methanol as a sole source of carbon and energy was isolated from the activated sludge. the bacteria are strictly aerobic gram-negative non-pigmented motile rods with a single polar flagellum. they do not form spores or capsules and have no complex intracytoplasmic membranes. they do not require vitamins and assimilate methanol via the hexulose phosphate pathway. the g+c content of dna is 51.0 +/- 0.5 mole%. the optimal temperature is 30-38 d ...19817219216
energy conservation in the terminal region of the respiratory chain of the methylotrophic bacterium methylophilus methylotrophus.reduced + co minus reduced difference spectra of respiratory membranes prepared from methanol-limited cultures of methylophilus methylotrophus confirm the presence of three co-binding cytochromes i.e. cytochromes aa3, o and cco. the kinetics of cyanide inhibition indicate that the respiratory chain of this organism is branched at the level of cytochrome c to two major terminal oxidases, viz. cytochromes aa3 and o; cytochrome cco is probably not a physiologically significant oxidase. determinatio ...19817285910
the primary structure of hyphomicrobium x dimethylamine dehydrogenase. relationship to trimethylamine dehydrogenase and implications for substrate recognition.the gene encoding dimethylamine dehydrogenase from hyphomicrobium x has been cloned and over-expressed in escherichia coli. using the chemically determined protein sequence, primers were designed to amplify dna fragments encoding the proximal and distal parts of the gene. these fragments were used to synthesise two probes and the dmd gene was cloned as part of two bamhi fragments isolated from digested genomic dna. the sequence of the complete open reading frame was determined on both strands an ...19957556160
mmei, a class-iis restriction endonuclease: purification and characterization.two restriction endonucleases, mmei and mmeii, from methylophilus methylotrophus were purified to homogeneity. both enzymes belong to the class-ii restriction endonucleases (enases) but exhibit very different enzymatic and physical properties. mmeii is a typical member of class-ii enases. it is a polymeric protein composed of 50-kda subunits. in contrast to mmeii, mmei is a monomeric protein of 101 kda, cleaving a dna molecule 20/18 nucleotides away from the asymmetric recognition sequence (5'-t ...19957607532
possible regulation of the methanol dehydrogenase fro methylophilus methylotrophus by its oxidized electron acceptor. 19807004951
the flavinylation reaction of trimethylamine dehydrogenase. analysis by directed mutagenesis and electrospray mass spectrometry.the flavinylation reaction products of wild-type and mutant forms of trimethylamine dehydrogenases purified from methylophilus methylotrophus (bacterium w3a1) and escherichia coli were studied by electrospray mass spectrometry (esms). the esms analyses demonstrated for the first time that wild-type enzyme expressed in m. methylotrophus is predominantly in the holoenzyme form, although a small proportion is present as the deflavo enzyme. esms demonstrated that the deflavo forms of the recombinant ...19957768915
the effect of edta and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, methylophilus methylotrophus.the effects of edta and related metal-chelating agents on the respiratory system of the methylotrophic bacterium methylophilus methylotrophus have been investigated. edta completely inhibited whole cell methanol oxidase activity and concomitantly decreased the aerobic steady-state reduction level of cytochrome c, but only partially inhibited methanol dehydrogenase activity. analysis of the inhibition kinetics of edta and other chelating agents on methanol oxidase activity indicated that they wer ...19846420156
genetic organization of the mau gene cluster in methylobacterium extorquens am1: complete nucleotide sequence and generation and characteristics of mau mutants.the nucleotide sequence of the methylamine utilization (mau) gene region from methylobacterium extorquens am1 was determined. open reading frames for 11 genes (maufbedacjglmn) were found, all transcribed in the same orientation. the maub, maua, and mauc genes encode the periplasmic methylamine dehydrogenase (madh) large and small subunit polypeptides and amicyanin, respectively. the products of maud, maug, maul, and maum were also predicted to be periplasmic. the products of mauf, maue, and maun ...19948021187
genetic mapping in methylophilus methylotrophus as1.prime plasmids have been isolated from methylophilus methylotrophus as1 using the incp-1 plasmid pmo172. such plasmids can complement mutant function when transferred to appropriate strains of pseudomonas aeruginosa pao. from the range of particular functions complemented by each prime plasmid, a preliminary map of the m. methylotrophus as1 genome with four groups of linked markers was obtained. physical examination of two of these prime plasmids showed that each had acquired an additional dna s ...19836308129
the terminal respiratory chain of the methylotrophic bacterium methylophilus methylotrophus.cytochrome oxidase o has been isolated from the obligately aerobic, methylotrophic bacterium methylophilus methylotrophus in the form of a cytochrome cl-o complex. the latter is comprised of cytochrome cl (mr 21 000) and cytochrome o (mr 29 000) in a 1-2:1 ratio, possibly in association with one or more minor polypeptides; the complex exhibits a high ascorbate-tmpd oxidase activity which is inhibited non-competitively by cyanide (ki approximately 2 microm). in contrast, the oxidation of methanol ...19836303840
an unusual conformation of the methionine haem ligand in cytochrome cl established by two-dimensional 1h-nmr.a complete relaxation-matrix analysis of noesy cross-peak intensities was used to determine the conformation of the methionine ligand to the haem group in two ferrocytochromes cl from methylophilus methylotrophus and methylobacterium extorquens, including the configuration at the sulphur. the conformation of the axial methionine is of a type reported only for the cytochromes c5 from pseudomonas mendocina and azotobacter vinelandii. although the conformation of the methionine is unusual, the para ...19948055954
a broad-host-range cloning vector transposable to various replicons.a system is described for the stable insertion of cloned dna sequences into the chromosomes of gram-negative bacteria. two broad-host-range plasmids form the basis of the system: one (the "carrier") contains a transposable dna sequence into which foreign dna can be cloned; the second (the "helper") provides transposition functions in trans. both plasmids can be readily transferred between gram-negative bacteria by conjugation. instability of the carrier allows enrichment for the products of tran ...19836301943
expression of a chemically synthesized human alpha 1 interferon gene.cells of escherichia coli containing a chemically synthesized human alpha 1 interferon (ifn-alpha 1) gene, under control of the lac promoter, make a product with biological properties indistinguishable from those of the natural ifn-alpha 1 [antiviral activity, acid stability, species crossreactivity, inactivation by antisera directed against leukocyte or namalwa cell interferon, and stimulation of (2'-5')oligoadenylate synthetase activity]. similar levels of ifn synthesis were obtained when the ...19826181502
on the dielectrically observable consequences of the diffusional motions of lipids and proteins in membranes. 2. experiments with microbial cells, protoplasts and membrane vesicles.the dielectric properties of suspensions of intact cells of methylophilus methylotrophus, paracoccus denitrificans and bacillus subtilis have been measured in the frequency range 1 khz to 13 mhz. all possess a pronounced dispersion corresponding in magnitude and relaxation time to the "beta-dispersion" in a terminology defined by schwan [adv. biol. med. phys. 5:147-209 (1957)]. the latter two strains, but not m. methylotrophus, also possess a substantial alpha-dispersion. the relaxation time of ...19853935420
transfer of plasmids from escherichia coli and pseudomonas aeruginosa to methylotrophic bacteria and their detection in the new hosts.several plasmids of incompatibility group p were transferred from escherichia coli and pseudomonas aeruginosa strains to methylophilus methylotrophus and two other methylotrophs to test their recipient ability. the presence of plasmids in transconjugants was confirmed by electrophoretic analysis. optimal conditions for detection of plasmid dna in the strains tested based on alkaline lysis of cells at elevated temperature were established. special behaviour of plasmids carrying the mu phage in me ...19873121477
identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium methylobacterium organophilum xx.a promoter-probe vector (phx200) was constructed using the broad-host-range cosmid pla2917 and a promoterless xyle gene of pseudomonas as the reporter gene. insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxf (methanol oxidation) in front of the xyle gene in phx200v-47 resulted in high-level expression of the xyle gene product--catechol 2,3-dioxygenase--in methylobacterium organophilum xx. the specific activity of the enzyme was four times higher in me ...19938515233
a high-potential soluble cytochrome c-551 from the purple phototrophic bacterium chromatium vinosum is homologous to cytochrome c8 from denitrifying pseudomonads.a minor cytochrome c-551 component of chromatium vinosum was previously found to efficiently couple electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center. we have now determined the amino acid sequence of this cytochrome c-551 and find that it is homologous to cytochrome c8 (formerly called pseudomonas cytochrome c-551). it is most similar to methylophilus methylotrophus, rhodocyclus tenuis, and azotobacter vinelandii cytochromes c8 (respectively, 57%, 52% a ...19968612646
physical, biochemical, and immunological characterization of a thermostable amidase from klebsiella pneumoniae nctr 1.an amidase capable of degrading acrylamide and aliphatic amides was purified to apparent homogeneity from klebsiella pneumoniae nctr 1. the enzyme is a monomer with an apparent molecular weight of 62,000. the ph and temperature optima of the enzyme were 7.0 and 65 degrees c, respectively. the purified amidase contained 11 5,5-dithiobis(2-nitrobenzoate) (dtnb)-titratable sulfhydryl (sh) groups. in the native enzyme 1.0 sh group readily reacted with dtnb with no detectable loss of activity. titrat ...19968636044
cytochrome c'' isolated from methylophilus methylotrophus. an example of bis-histidine-co-ordinated fe3+ haem, with near-perpendicular orientation of the ligands.cytochrome c'' (methylophilus methylotrophus) is a soluble protein, mr 15,000, possessing one haem which is high-spin in the reduced state but switches to a low-spin form on oxidation. low-temperature electron-paramagnetic-resonance spectroscopy of the oxidized state shows a low-spin signal at gz = 3.65 with a folded line-shape typical of a haem of low rhombicity, and the near-infrared magnetic-circular-dichroism (m.c.d.) spectra reveal an unusually intense (delta epsilon = 400 m-1.cm-1 at 5 t, ...19902169241
protein engineering studies of dichloromethane dehalogenase/glutathione s-transferase from methylophilus sp. strain dm11. ser12 but not tyr6 is required for enzyme activity.the structural gene for dichloromethane dehalogenase/glutathione s-transferase (gst, ec 2.5.1.18) from methylophilus sp. strain dm11 was subcloned into a multicopy plasmid under the control of the t7 polymerase promoter, allowing expression in escherichia coli and easy purification of the enzyme in good yield. several point mutations leading to amino acid changes at residues tyr6, his8 and ser12 of the protein were introduced in this gene. mutations at tyr6, the n-terminal tyrosine known to be e ...19968706748
preparation of isotopically labeled ribonucleotides for multidimensional nmr spectroscopy of rna.a general method for large scale preparation of uniformly isotopically labeled ribonucleotides and rnas is described. bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. these are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare rnas for nmr studies. for 15n-labeling, e.coli is grown on 15n-ammonium sulfate, whereas for 13c-labeling, methylophilus methylotrophu ...19921383928
the three-dimensional structures of methanol dehydrogenase from two methylotrophic bacteria at 2.6-a resolution.the structures of methanol dehydrogenase (medh) from two closely related methylotrophic bacteria, methylophilus methylotrophus and w3a1, have been determined at 2.6-a resolution. the molecule, a quinoprotein of molecular mass of about 138 kda, contains two heavy (h) and two light (l) subunits of unknown sequence and two molecules of noncovalently associated pyrroloquinoline quinone. the two enzymes crystallize isomorphously in space group p2(1) with one h2l2 heterotetramer in the asymmetric unit ...19921331050
transformation of methylotrophic bacteria by electroporation.an efficient system for electroporation of the methylotrophic bacteria hyphomicrobium facilis, hyphomicrobium denitrificans, methylobacillus glycogenes, methylobacterium extorquens, and methylophilus methylotrophus is described. it could be demonstrated that vectors based on the broad-host-range plasmid pbbr1 could be transferred into these strains. plasmid pbbr1kan (3.9 kb), a kanamycin-resistant derivative of pbbr1, was suitable for transformation experiments in these methylotrophic bacteria. ...19979090108
the interaction of methanol dehydrogenase and its electron acceptor, cytochrome cl in methylotrophic bacteria.the interactions of methanol dehydrogenase (mdh, ec1.1.99.8) with its specific electron acceptor cytochrome cl has been investigated in methylobacterium extorquens and methylophilus methylotrophus. the mdhs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these mdhs with their specific electron acceptor, cytochrome cl, has been studied using a novel assay system. electrostatic reactions are involved in 'docking' of the two proteins. edta inhibi ...19921311606
an outer-membrane porin inducible by short-chain amides and urea in the methylotrophic bacterium methylophilus methylotrophus.the fmda and fmdb genes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacterium methylophilus methylotrophus were recloned with additional flanking dna (psw1). fmdc, encoding a weakly hydrophilic protein containing an n-terminal signal sequence, was identified upstream of fmdab. the derived amino acid sequence of mature fmdc (m(r) 39204) showed that it was rich in beta-sheet and aromatic amino acids, and exhibited significant similarities to severa ...19979245819
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 o(2)-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 co ...19807236221
identification of a novel determinant of glutathione affinity in dichloromethane dehalogenases/glutathione s-transferases.bacterial dichloromethane dehalogenases catalyze the glutathione-dependent hydrolysis of dichloromethane to formaldehyde and are members of the enzyme superfamily of glutathione s-transferases involved in the detoxification of electrophilic compounds. numerous protein engineering studies have addressed questions pertaining to the substrate specificity, the reaction mechanism, and the kinetic pathway of glutathione s-transferases. in contrast, the molecular determinants for binding of the glutath ...19979299530
purification and properties of the methanol dehydrogenase from methylophilus methylotrophus.1. a dye-linked methanol dehydrogenase, resembling many others from a variety of methylotrophic bacteria, was purified to homogeneity from extracts of methanol-grown methylophilus methylotrophus. 2. the enzyme was very stable in the presence of methanol; in the absence of methanol it had a half-life of 1-2 days at 4 degrees c. 3. the value of a1% 1cm,280 was 17.5. 4. the enzyme retained bound methanol after passage through sephadex g-25. this tightly-bound methanol slowly exchanged with free [14 ...19816802134
the autoreducible cytochromes c of the methylotrophs methylophilus methylotrophus and pseudomonas am1.the two types of soluble cytochrome c (cytochrome ch and cytochrome cl) found in methylotrophs are completely distinct proteins; one type is not a dimer or degradation product of the other. free thiol groups are probably not involved in the unusually rapid autoreduction of the cytochromes at high ph. the axial ligands to the haem iron, histidine and methionine, are the same as in other low-spin cytochromes c. the methionine ligand is displaced at high ph by an alternative strong-field ligand. th ...19826295363
the purification and properties of the soluble cytochromes c of the obligate methylotroph methylophilus methylotrophus.the obligate methylotroph methylophilus methylotrophus contains three distinct soluble cytochromes c. the major cytochromes, cytochrome ch (about 50% of the total) and cytochrome cl (about 42%), were similar in most respects to the cytochromes ch and cl of the facultative methylotroph pseudomonas am1 [o'keeffe & anthony (1980) biochem. j. 192, 411-419]. cytochrome ch had a high isoelectric point, a midpoint redox potential at ph 7.0 of 373 mv and a low molecular weight (8500). the cytochrome cl ...19806263254
effects of bacterial host and dichloromethane dehalogenase on the competitiveness of methylotrophic bacteria growing with dichloromethane.methylobacterium sp. strain dm4 and methylophilus sp. strain dm11 can grow with dichloromethane (dcm) as the sole source of carbon and energy by virtue of homologous glutathione-dependent dcm dehalogenases with markedly different kinetic properties (the kcat values of the enzymes of these strains are 0.6 and 3.3 s-1, respectively, and the km values are 9 and 59 microm, respectively). these strains, as well as transconjugant bacteria expressing the dcm dehalogenase gene (dcma) from dm11 or dm4 on ...19989546153
homology model of the quinohaemoprotein alcohol dehydrogenase from comamonas testosteroni.a molecular model of qh-adh, the quinohaemoprotein alcohol dehydrogenase from comamonas testosteroni, has been built by homology modelling. sequence similarity of n-terminal residues 1-570 with the alpha-subunit of quinoprotein methanol dehydrogenases (mdhs) from methylophilus methylotrophus w3a1 and methylobacterium extorquens provided a basis for the design of the pqq-binding domain of qh-adh. minimal sequence similarity with cytochrome c551 from ectothiorhodospira halophila and cytochrome c5 ...19989613842
the apparent oxidation of nadh by whole cells of the methylotrophic bacterium methylophilus methylotrophus. a cautionary tale.previous reports that whole cells of methylophilus methylotrophus oxidase exogenous nadh have been investigated. essentially identical rates of oxygen consumption were observed following the addition of methanol or nadh to whole cells. both activities were inhibited by edta and hydroxylamine, but not by hqno, and exhibited similar ph optima. analyses of the reaction stoichiometry with nadh as substrate showed that the expected amount of oxygen was consumed, but also revealed acidification (inste ...19863098164
isolation and computer-aided characterization of mmei, a type ii restriction endonuclease from methylophilus methylotrophus.a type ii restriction endonuclease, mmei, has been purified from the obligate methylotroph, methylophilus methylotrophus. the enzyme was shown to have the non-palindromic recognition sequence 5'-t c c pu a c (n)20-3', 3'-a g g py t g (n)18-5' and to cleave (as indicated) on the 3' side, generating a two nucleotide 3' projection. determination of the recognition sequence was achieved using two new computer programs; recog, which predicts recognition sequences from the pattern of restriction fragm ...19863016643
unusual redox properties of electron-transfer flavoprotein from methylophilus methylotrophus.the most positive redox potential ever recorded for a flavin adenine dinucleotide (fad) containing protein has been measured for an electron-transfer flavoprotein (etf) synthesized by methylophilus methylotrophus. this potential value, 0.196 v versus the standard hydrogen electrode (vs she), was measured at ph 7.0 for the one-electron reduction of fully oxidized etf (etfox) to the red anionic semiquinone form of etf (etf.-). quantitative formation of etf.- was observed. the first successful redu ...19892605209
the periplasmic modifier protein for methanol dehydrogenase in the methylotrophs methylophilus methylotrophus and paracoccus denitrificans.a modifier protein (m-protein), which increases the affinity of methanol dehydrogenase (mdh) for alcohols but decreases its affinity for formaldehyde, has been partially purified from methylophilus methylotrophus and paracoccus denitrificans. analysis was complicated by non-protein factors in bacterial extracts that are able to mimic m-protein in one of its functions-that of increasing the activity of mdh with butane-1,3-diol in the dye-linked assay system. the 67 kda polypeptide, previously ide ...19911663153
involvement of a labile axial histidine in coupling electron and proton transfer in methylophilus methylotrophus cytochrome c''.methylophilus methylotrophus cytochrome c'' is an unusual monohaem protein (15 kda) undergoing a redox-linked spin-state transition [santos, h. & turner, d. l. (1988) biochim. biophys. acta 954, 277-286]. the midpoint redox potential of cytochrome c" was measured over the ph range 4-10. the ph dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the prote ...19921325909
cloning, sequencing, and mutation of a gene for azurin in methylobacillus flagellatum kt.the gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium methylobacillus flagellatum kt. partial sequence data showed that the organization of these genes was similar to that found in methylophilus methylotrophus w3a1-ns, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in m. flagellatum kt. however, a gene encoding azurin was discovered at the 3' end of the mau ...19957635847
cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium w3a1 electron transfer flavoprotein.the genes encoding the two different subunits of the electron transfer flavoprotein (etf) from the methylotrophic bacterium w3a1 have been identified, cloned, and sequenced. a 0.8-kilobase pair dna fragment was generated for use as a molecular probe by the amplification of genomic sequences using the polymerase chain reaction and a primer pair with degenerate sequences derived from the nh2-terminal amino acid sequences determined for the etf subunits purified from w3a1. the screening of a partia ...19947798207
improved conversion of methanol to single-cell protein by methylophilus methylotrophus.the glutamate dehydrogenase gene of escherichia coli has been cloned into broad host-range plasmids and can complement glutamate synthase mutants of methylophilus methylotrophus. assimilation of ammonia via glutamate dehydrogenase is more energy-efficient than via glutamate synthase, thus the recombinant organism converts more growth substrate, methanol, into cellular carbon.19806776410
crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydrogenases (adhs): a soluble monomeric adh from pseudomonas putida hk5 (adh-iib) and a heterotrimeric membrane-bound adh from gluconobacter suboxydans (adh-gs).crystals of a soluble monomeric quinocytochrome alcohol dehydrogenase (adh-iib) and of a trimeric membrane-associated quinocytochrome alcohol dehydrogenase (adh-gs) have been obtained. the adh-iib crystals are triclinic, with one monomer in the unit cell, and were obtained in the presence of peg 8000, sodium citrate, hepes buffer and 2-propanol. x-ray data were collected at 110 k to 1. 9 a resolution (r(merge) = 6.4%) and the orientation of a methanol dehydrogenase search molecule (from methylop ...199910531500
determination of the gene sequence and the three-dimensional structure at 2.4 angstroms resolution of methanol dehydrogenase from methylophilus w3a1.the dna sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from methylophilus methylotrophus w3a1 have been determined. the deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 angstrom resolution against x-ray data collected on a hamlin area detector. the structure was refined using the programs profft and x-plor with several model building step interspersed. the final model contains two heavy chains (571 amino acids), two ...19968676383
crystallization and preliminary crystallographic investigation of electron-transfer flavoprotein from the bacterium methylophilus w3a1.electron-transfer flavoprotein from methylophilus w3a1 has been crystallized using the sitting drop vapour diffusion technique. hexagonal crystals, suitable for high-resolution structure determination grow to approximately 0.7 mm x 0.7 mm x 0.5 mm in size and diffract to at least 2.2 a. the space group is either p6(1) or p6(5) with unit cell dimensions a,b = 119.1 a and c = 85.6 a.19948028009
molecular cloning of a reca-like gene of methylophilus methylotrophus and identification of its product.a recombinant plasmid, ppf5, has been constructed which contains a 5.5-kb fragment of methylophilus methylotrophus dna inserted into pat153, and which confers resistance to uv and mitomycin c (mc) upon escherichia coli reca mutants. hybridisation analysis indicates that sequence homology exists between the cloned dna and a fragment of e. coli chromosomal dna that contains the reca gene. tn1000 insertions have been isolated within ppf5 that inactivate its ability to complement reca mutations, and ...19863021589
formation of w(3)a(1) electron-transferring flavoprotein (etf) hydroquinone in the trimethylamine dehydrogenase x etf protein complex.the electron-transferring flavoprotein (etf) from methylophilus methylotrophus (sp. w(3)a(1)) exhibits unusual oxidation-reduction properties and can only be reduced to the level of the semiquinone under most circumstances (including turnover with its physiological reductant, trimethylamine dehydrogenase (tmadh), or reaction with strong reducing reagents such as sodium dithionite). in the present study, we demonstrate that etf can be reduced fully to its hydroquinone form both enzymatically and ...200010777543
cytochrome c" from the obligate methylotroph methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph rhodobacter sphaeroides.the complete primary structure of an unusual soluble cytochrome c isolated from the obligate methylotrophic bacterium methylophilus methylotrophus has been determined to contain 124 amino acids and to have an average molecular mass of 14293.0 da. the sequence has two unusual features: firstly, the location of the heme-binding cysteines is far downstream from the n-terminus, namely at positions 49 and 52; secondly, an extra pair of cysteine residues is present near the c-terminus. in both respect ...199910354493
reaction of rat liver glutathione s-transferases and bacterial dichloromethane dehalogenase with dihalomethanes.dichloromethane dehalogenase from methylophilus sp. dm11 is a glutathione s-transferase homolog that is specifically active with dihalomethane substrates. this bacterial enzyme and rat liver glutathione s-transferases were purified to investigate their relative reactivity with ch2cl2 and related substrates. rat liver alpha class glutathione transferases were inactive and mu class enzymes showed low activity (7-23 nmol/min/mg of protein) with ch2cl2. theta class glutathione transferase 5-5 from r ...19948132617
isolation and characterization of the methylophilus sp. strain dm11 gene encoding dichloromethane dehalogenase/glutathione s-transferase.the restricted facultative methylotroph methylophilus sp. strain dm11 utilizes dichloromethane as the sole carbon and energy source. it differs from other dichloromethane-utilizing methylotrophs by faster growth on this substrate and by possession of a group b dichloromethane dehalogenase catalyzing dechlorination at a fivefold-higher rate than the group a enzymes of slow-growing strains. we isolated dcma, the structural gene of the strain dm11 dichloromethane dehalogenase, to elucidate its rela ...19948206823
characterisation of a binding-protein-dependent, active transport system for short-chain amides and urea in the methylotrophic bacterium methylophilus methylotrophus.three genes (fmdcab) encoding an outer-membrane porin for short-chain amides and urea, formamidase, and a putative regulatory protein in methylophilus methylotrophus have previously been cloned and characterised. three genes have now been identified downstream of fmdb, viz fmdd encoding a hydrophilic protein containing an n-terminal signal sequence, and fmdef encoding hydrophobic transmembrane proteins. the derived amino acid sequence of mature fmdd (predicted molecular mass 41,870 da) was simil ...19989492267
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 oxidatio ...19816272739
haloacetonitriles are low k1 inhibitors of bacterial dichloromethane dehalogenases.distinct dichloromethane dehalogenases from methylobacterium sp. strain dm4 and methylophilus dm11 were inhibited by low concentrations of haloacetonitriles. chloroacetonitrile (clch2cn) showed maximal inhibition at a stoichiometry of 1 mol inhibitor:1 mol holoenzyme for both enzymes. this stoichiometry is suggestive of one active site per holoenzyme or extreme negative cooperativity amongst the subunits. radiolabelled clch2cn dissociated completely or partially from the two dehalogenases, respe ...19938267624
characterization of the haem environment in methylophilus methylotrophus ferricytochrome c" by 1h-nmr.two-dimensional nmr techniques have been used to assign proton resonances in the haem cavity of methylophilus methylotrophus cytochrome c", a monohaem protein with bis-histidinyl ligation which has been shown to couple electron and proton transfer. all the assignments were made directly for the oxidized paramagnetic form of the cytochrome. nearly all of the haem protons (90%) and the protons of both axial ligands have been assigned; the side-chain protons from four other residues in the haem poc ...19938394812
enzymatic h-transfer requires vibration-driven extreme tunneling.enzymatic breakage of the substrate c-h bond by methylophilus methyltrophus (sp. w3a1) methylamine dehydrogenase (madh) has been studied by stopped-flow spectroscopy. the rate of reduction of the tryptophan tryptophylquinone (ttq) cofactor has a large kinetic isotope effect (kie = 16.8 +/- 0.5), and the kie is independent of temperature. analysis of the temperature dependence of c-h bond breakage revealed that extreme (ground state) quantum tunneling is responsible for the transfer of the hydrog ...199910074378
sequence variation in dichloromethane dehalogenases/glutathione s-transferases.dichloromethane dehalogenase/glutathione s-transferase allows methylotrophic bacteria to grow with dichloromethane (dcm), a predominantly man-made compound. bacteria growing with dcm by virtue of this enzyme have been readily isolated in the past. so far, the sequence of the dcma gene encoding dcm dehalogenase has been determined for methylobacterium dichloromethanicum dm4 and methylophilus sp. dm11. dcm dehalogenase genes closely related to that of strain dm4 were amplified by pcr and cloned fr ...200111238968
molecular characterisation of formamidase from methylophilus methylotrophus.a 3.2-kbp psti fragment of dna encoding formamidase from the methylotrophic bacterium methylophilus methylotrophus which had previously been cloned (pnw3) [wyborn, n.r., scherr, d.j. & jones, c.w. (1994) microbiology 140, 191-195], was subcloned as a 2.3 kbp hindiii fragment (pnw323). nucleotide sequencing showed that the subclone contained two genes which encoded formamidase (fmda) and a possible regulatory protein (fmdb). predicted molecular masses for fmda and fmdb were 44438 da (compared wit ...19968841393
the reaction of trimethylamine dehydrogenase with trimethylamine.the reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the ph range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. as in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. the observed rate constant for the fast phase e ...199910224069
creating auxotrophic mutants in methylophilus methylotrophus as1 by combining electroporation and chemical mutagenesis.stable auxotrophic mutants of the methylotroph methylophilus methylotrophus as1 were obtained by a novel mutagenesis technique in which electroporation is used to transport the chemical mutagen n-methyl-n'-nitro-n-nitrosoguanidine (mnng) across the cell membrane. by combining chemical mutagenesis with electroporation and screening single colonies for auxotrophy in 36 different amino acids and growth factors, 3 auxotrophs per 156 colonies screened were obtained, whereas no auxotrophs were found w ...19979274053
cloning and analysis of methanol oxidation genes in the methylotroph hyphomicrobium methylovorum gm2.the gene encoding the alpha-subunit of methanol dehydrogenase (mxaf) and its flanking region was isolated from a methylotrophic bacterium, hyphomicrobium methylovorum gm2. the deduced amino acid sequence of mxaf showed 80, 80, 74 and 66% identity with those of methylobacterium extorquens am1, m. organophilum xx, paracoccus denitrificans and methylophilus methylotrophus, respectively. the putative mxaf promoter sequence (-35 -aaagaca-, -10 -tagaa-) observed in other methylotrophs was not found in ...19979311140
cloning and sequence analysis of the gene encoding methylophilus methylotrophus cytochrome c", a unique protein with a perpendicular orientation of the histidinyl ligands.cytochrome c" from methylophilus methylotrophus is an unusual monohaem protein that undergoes a major redox-linked spin-state transition: one of the two axial histidines bound to the iron in the oxidised form is detached upon reduction and a proton is taken up. a 3.5-kb dna fragment, containing the gene encoding cytochrome c" (cyca), has been cloned and sequenced. the cytochrome c" gene codes for a pre-protein with a typical prokaryotic 20-residue signal sequence, suggesting that the protein is ...199910524262
organization of the methylamine utilization (mau) genes in methylophilus methylotrophus w3a1-ns.the organization of genes involved in utilization of methylamine (mau genes) was studied in methylophilus methylotrophus w3a1. the strain used was a nonmucoid variant termed ns (nonslimy). the original mucoid strain was shown to be identical to the ns strains on the basis of chromosomal digest and hybridization patterns. an 8-kb psti fragment of the chromosome from m. methylotrophus w3a1-ns encoding the mau genes was cloned and a 6,533-bp region was sequenced. eight open reading frames were foun ...19948021188
x-ray scattering studies of methylophilus methylotrophus (sp. w3a1) electron-transferring flavoprotein. evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase.small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (etf) from methylophilus methylotrophus (sp. w(3)a(1)). analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. comparison of the molecular envelope of etf from the x-ray scattering data with pr ...200010766748
identification of klebsiella pneumoniae genes involved in intestinal colonization and adhesion using signature-tagged mutagenesis.klebsiella pneumoniae is an opportunistic pathogen responsible for nosocomial infections that initially colonize the intestinal tract of patients. signature-tagged mutagenesis was used to identify genes required for this function. a library of 2,200 mutants was analyzed for the inability of the mutants to survive in a murine model of intestinal colonization and to adhere to human intestinal cells (int-407) in vitro. twenty-nine attenuated mutants were selected for further analyses after competit ...200212117993
two intertwined methylation activities of the mmei restriction-modification class-iis system from methylophilus methylotrophus.the class-iis restriction endonuclease, r.mmei, was isolated from methylophilus methylotrophus. it was originally described as a monomeric enzyme, with the native mr 105000+/-7000, which did not cleave dna efficiently [boyd et al. (1986) nucleic acids res. 14, 5255-5274; tucholski et al. (1995) gene 157, 87-92]. however, it was discovered that r.mmei endonucleolytic activity is enhanced by s-adenosyl-l-methionine (adomet) and sinefungin, an analogue of adomet. surprisingly, the purified r.mmei e ...19989858752
detailed active site configuration of a new crystal form of methanol dehydrogenase from methylophilus w3a1 at 1.9 a resolution.the three-dimensional structure of a new crystal form of methanol dehydrogenase from methylophilus w3a1 has been obtained in the presence of substrate using data recorded at a synchrotron. the structure of this approximately 140 kda heterotetramer, refined at 1. 9 a resolution, reveals the detailed configuration of its redox cofactor, pyrroloquinoline quinone (pqq). c4, one of the oxygen-bearing atoms of this orthoquinone is in a planar configuration while c5, which bears the other quinone oxyge ...19999930981
crystal structures of an oxygen-binding cytochrome c from rhodobacter sphaeroides.the photosynthetic bacterium rhodobacter sphaeroides produces a heme protein (shp), which is an unusual c-type cytochrome capable of transiently binding oxygen during autooxidation. similar proteins have not only been observed in other photosynthetic bacteria but also in the obligate methylotroph methylophilus methylotrophus and the metal reducing bacterium shewanella putrefaciens. a three-dimensional structure of shp was derived using the multiple isomorphous replacement phasing method. besides ...200010821858
enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione s-transferases.the kinetic properties of bacterial and rat liver glutathione s-transferases (gst) active with dichloromethane (dcm) were compared. the theta class glutathione s-transferase (rgstti-1) from rat liver had an affinity for dihalomethanes lower by three orders of magnitude (k(app) > 50 mm) than the bacterial dcm dehalogenase/gst from methylophilus sp. dm11. unlike the bacterial dcm dehalogenase, the rat enzyme was unable to support growth of the dehalogenase minus methylobacterium sp. dm4-2cr mutant ...199910350186
purification and characterization of the mutant enzyme w117y of the dichloromethane dehalogenase from methylophilus sp. strain dm11. 199810075637
radiolytic studies of trimethylamine dehydrogenase. spectral deconvolution of the neutral and anionic flavin semiquinone, and determination of rate constants for electron transfer in the one-electron reduced enzyme.trimethylamine dehydrogenase from the pseudomonad methylophilus methylotrophus has been examined using the technique of pulse radiolysis to rapidly introduce a single reducing equivalent into the enzyme. using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with ferricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low ph, respectively, of the unique 6-cy ...200010859304
ph dependence of structural and functional properties of oxidized cytochrome c" from methylophilus methylotrophus.cytochrome c" from methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up. the kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c" have been investigated over the ph range between 1.4 and 10.0. the rate of reduction displays proton-linked transitions ...19979312076
use of stable-isotope probing, full-cycle rrna analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community.a denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (sbr) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. the sbr was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. the sbr denitrification rate improved from less than 0.02 mg of no(3)(-)-n mg of mixed-liquor volatile suspended solids (mlvss)(-1) h(-1) ...200414711691
solution structure and metal-ion binding of the p4 element from bacterial rnase p rna.we determined the solution structure of two 27-nt rna hairpins and their complexes with cobalt(iii)-hexammine (co(nh3)3+(6)) by nmr spectroscopy. the rna hairpins used in this study are the p4 region from escherichia coli rnase p rna and a c-to-u mutant that confers altered divalent metal-ion specificity (ca2+ replaces mg2+) for catalytic activity of this ribozyme. co(nh3)3+(6) is a useful spectroscopic probe for mg(h2o)2+(6)-binding sites because both complexes have octahedral symmetry and have ...200010999599
[deuterium oxide as a stress factor to the methylotrophic bacterium methylophilus sp].the adaptation of the methylotrophic bacterium methylophilus sp. b-7741 to growth in highly deuterated media was studied. for the first time, we showed the cross adaptation of bacterial cells to deuterated media and oxidative and osmotic stresses. the activity at catalase in deuterated cells was higher than in the control cells. deuterated cell-free culture liquids showed protective effects on the growth of methylophilus sp. b-7741 in deuterated media, which was manifested as an increase in the ...200415198027
solution structure of methylophilus methylotrophus cytochrome c": insights into the structural basis of haem-ligand detachment.cytochrome c" from methylophilus methylotrophus is a monohaem protein with 124 amino acid residues. the iron has two histidine ligands in the oxidised form, one of which detaches and picks up a proton when the protein is reduced. thus, both forms are paramagnetic. the structure of the oxidised form in solution, determined from nmr data is presented. the family of structures has an average backbone rmsd value of 0.53 a, and a heavy atom rmsd value of 0.95 a, within a target function range of 32 % ...200111327772
knowledge-based modeling of a bacterial dichloromethane dehalogenase.a three-dimensional structural model of the dichloromethane dehalogenase (dcmd) from methylophilus sp. dm11 is constructed based on sequence similarities to the glutathione s-transferases (gsts). to maximize sequence identity and minimize gaps in the alignment, a hybrid approach is used that takes advantage of the increased homology found between dm11 and domain i of the sheep blowfly theta class gst (residues 1-79) and domain ii of the human alpha class gst (residues 81-222). the resulting stru ...19979188739
detection of ectoin biosynthesis genes in halotolerant aerobic methylotrophic bacteria. 200415378927
x-ray structure of the quinoprotein ethanol dehydrogenase from pseudomonas aeruginosa: basis of substrate specificity.the homodimeric enzyme form of quinoprotein ethanol dehydrogenase from pseudomonas aeruginosa atcc 17933 crystallizes readily with the space group r3. the x-ray structure was solved at 2.6 a resolution by molecular replacement. aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from methylobacterium extorquens or methylophilus w3a1. eight w-shaped beta-sheet motifs are arranged circularly in a propeller ...200010736230
[phylogenetic analysis of aerobic methylotrophic bacteria, using dichloromethane].the phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different c1-assimilation pathways was determined based on 16s ribosomal rna sequences and dna-dna hybridization data. the restricted facultative methylotroph "methylophilus leisingerii" dm11 with the ribulose monophosphate pathway was found to belong to the genus methylophilus cluster of the beta subdivision of the phylogenetic kingdom proteobacteria. the facultative methylotroph methylorhabdus multiv ...200111338843
histamine dehydrogenase from rhizobium sp.: gene cloning, expression in escherichia coli, characterization and application to histamine determination.the gene encoding histamine dehydrogenase in rhizobium sp. 4--9 has been cloned and overexpressed in escherichia coli. the coding region of the gene was 2,079 bp and encoded a protein of 693 amino acids with a calculated molecular mass of 76,732 da. this histamine dehydrogenase was related to histamine dehydrogenase from nocardioides simplex (54.5% identical), trimethylamine dehydrogenase from methylophilus methylotrophus (39.3% identical) and dimethylamine dehydrogenase from hyphomicrobium x (3 ...200515964650
methylophilus quaylei sp. nov., a new aerobic obligately methylotrophic bacterium.a new obligately methylotrophic bacterium (strain mtt) with the ribulose monophosphate pathway of carbon assimilation is described. the isolate, utilizing only methanol, is an aerobic, gram-negative, asporogenous, non-motile short rod multiplying by binary fission. its cellular fatty acids profile consists primarily of straight-chain saturated c16:0 and unsaturated c16:l acids. the major ubiquinone is q-8. the dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. diphosph ...200515997702
a mechanism for substrate-induced formation of 6-hydroxyflavin mononucleotide catalyzed by c30a trimethylamine dehydrogenase.experiments are described to determine the origin of the 6-hydroxyl group of 6-hydroxyfmn produced by the substrate-induced transformation of fmn in the c30a mutant of trimethylamine dehydrogenase. the conversion of fmn to 6-hydroxyfmn is carried out in the presence of h(2)(18)o and 18o(2), and the results clearly show that the 6-hydroxyl group is derived from molecular oxygen and not from water.200314592522
detection of potentially novel bacterial components of the human skin microbiota using culture-independent molecular profiling.although the micro-organisms forming the cutaneous microbiota are considered to play important roles in the modification and prevention of skin diseases, a comprehensive analysis of their composition has not yet been carried out because of difficulties in determining yet-to-be-cultured micro-organisms in the samples. swab-scrubbed forehead skin samples of five healthy volunteers were analysed by profiling 16s rrna genes, as well as by conventional culture methods, to provide a profile of the cut ...200516278439
inactivation of c30a trimethylamine dehydrogenase by n-cyclopropyl-alpha-methylbenzylamine, 1-phenylcyclopropylamine, and phenylhydrazine.trimethylamine dehydrogenase (tmadh) from the bacterium methylophilus methylotrophus (sp. w(3)a(1)) and its c30a mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine, n-cyclopropyl-alpha-methylbenzylamine, and 1-phenylcyclopropylamine. all three compounds irreversibly inactivated both the wild-type and c30a mutant enzymes, although phenylhydrazine was 10 times more potent than n-cyclopropyl-alpha-methylbenzylamine, which was much more potent than 1- ...200111456490
stable carbon isotope fractionation during degradation of dichloromethane by methylotrophic bacteria.stable carbon isotope fractionation during dichloromethane (dcm) degradation by methylotrophic bacteria was investigated under aerobic and nitrate-reducing conditions. the strains studied comprise several hyphomicrobium strains, methylobacterium, methylopila, methylophilus and methylorhabdus spp. that are considered to degrade dcm by a glutathione (gsh)-dependent dehalogenase enzyme system in the initial step. the stable carbon isotope fractionation factors (alphac) of the strains varied under a ...200616343330
isolation of auxotrophic mutants of methylophilus methylotrophus by modified-marker exchange.a method for stabilizing a transposon (tn5) has been developed which allows the isolation of stable auxotrophic mutants of methylophilus methylotrophus asi. insertion of tn5 into a cloned m. methylotrophus asi dna fragment encoding anthranilate synthase followed by transfer of the vector with the modified trpe gene to m. methylotrophus asi resulted in unstable auxotrophs among the recombinants. deletion of is50r, which encodes transposase production from tn5, stabilized the transposon after mobi ...198816347531
a novel directly coupled gradostat.the original bidirectional compound chemostat (gradostat) described by lovitt and wimpenny has been simplified by making a more compact apparatus in which chemical gradients are established by diffusion between adjacent culture chambers. the experimental model (diffusion coupled (dc) gradostat) consisted of five chambers whose contents could be agitated by turbines rotating in the horizontal plane on a common shaft. two biological experiments were designed to reveal the value of the dc gradost ...199211540058
community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms.the bacterial community structure, in situ spatial distributions and activities of nitrifying and denitrifying bacteria in biofilms treating industrial wastewater were investigated by combination of the 16s rrna gene clone analysis, fluorescence in situ hybridization (fish) and microelectrodes. these results were compared with the nitrogen removal capacity of the industrial wastewater treatment plant (iwtp). both nitrification and denitrification occurred in the primary denitrification (pd) tank ...200616477661
l-lysine biosynthetic pathway of methylophilus methylotrophus and construction of an l-lysine producer.previously, we showed that the enzymes aspartokinase (ak) and dihydrodipicolinate synthase (ddps), which are involved in l-lysine biosynthesis in the gram-negative obligate methylotroph methylophilus methylotrophus as1, were inhibited by allosteric effectors, including l-lysine. to elucidate further the regulation of l-lysine biosynthesis in m. methylotrophus, we cloned the genes encoding three other enzymes involved in this pathway, l-aspartate-beta-semialdehyde dehydrogenase, dihydrodipicolina ...200616483680
[effect of deuteration on methanol dehydrogenase activity in methylophilus sp. b-7741].we studied the effect of deuterium oxide present in the medium on the activity of methanol dehydrogenase (ec 1.1.99.8) from methylotrophic bacteria methylophilus sp. b-7741. methanol dehydrogenase activity in extracts of the biomass obtained in a highly deuterated medium (2h-enzyme) was 34-47% of enzyme activity in the control biomass, which depended on reaction conditions. the isotopic effects of substrate deuterium (methanol) for 1h-enzyme and 2h-enzyme were 1.37 +/- 0.05 and 1.38 +/- 0.01, re ...200415029692
identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing.stable-isotope probing (sip) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. a sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [(13)c]acetate or [(13)c]methanol as the main carbon source and nitrate as the electron acceptor. we analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon ...200616897304
enzymes of dimethylsulfone metabolism and the phylogenetic characterization of the facultative methylotrophs arthrobacter sulfonivorans sp. nov., arthrobacter methylotrophus sp. nov., and hyphomicrobium sulfonivorans sp. nov.novel methylotrophic arthrobacter and hyphomicrobium species are described. constitutive membrane-associated dimethylsulfone- and dimethylsulfoxide-reductases were found in arthrobacter methylotrophus strain tga and hyphomicrobium sulfonivorans strain s1. enzyme activities increased during growth with dimethylsulfone or dimethylsulfoxide, respectively, and different ratios of activity with different growth substrates indicated that they are separate enzymes. sds-page showed some membrane-associa ...200211807567
[site-directed mutagenesis of the dichloromethane dehalogenase gene from methylophilus sp. strain dm11].in order to investigate the role of different residues of methylophilus sp. strain dm11 dichloromethane dehalogenase for substrate binding, glutathione affinity, and catalytic activity, site-directed mutagenesis studies of the gene encoding the enzyme were carried out. the conserved tryptophane residue at 103 region was respectively substituted by phenylalanine, valine or asparagine. the conserved arginine residue at 109 region was substituted by leucine. the conserved tryptophane residue at 117 ...199812549326
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