Publications

genomic analyses of transport proteins in ralstonia metallidurans.	ralstonia (wautersia, cupriavidus) metallidurans (rme) is better able to withstand high concentrations of heavy metals than any other well-studied organism. this fact renders it a potential agent of bioremediation as well as an ideal model organism for understanding metal resistance phenotypes. we have analysed the genome of rme for genes encoding homologues of established and putative transport proteins; 13% of all genes in rme encode such homologues. nearly one-third of the transporters identi ...	2005	18629293
atomic force microscopic observation of in vitro polymerized poly[(r)-3-hydroxybutyrate]: insight into possible mechanism of granule formation.	atomic force microscopy (afm) was used to study the formation and growth of poly[(r)-3-hydroxybutyrate] (phb) structures formed in the enzymatic polymerization of (r)-3-hydroxybutyryl coenzyme a [(r)-3-hbcoa] in vitro. poly(3-hydroxyalkanoate) (pha) synthase (phac(re)) from ralstonia eutropha, a class i synthase, was purified by one-step purification and then used for in vitro reactions. before the reaction, phac(re) molecules were deposited on highly oriented pyrolytic graphite (hopg) and obser ...	2005	16153105
secondary transporters for nickel and cobalt ions: theme and variations.	nickel/cobalt transporters (nicots), a family of secondary metal transporters in prokaryotes and fungi, are characterized by an eight-transmembrane-domain (tmd) architecture and mediate high-affinity uptake of cobalt and/or nickel ions into the cells. one of the strongly conserved regions within the nicots is the signature sequence rha(v/f)dadhi within tmd ii. this stretch of amino acid residues plays an important role in the affinity, velocity and specificity of metal transport. some relatives ...	2005	16158232
control of expression of a periplasmic nickel efflux pump by periplasmic nickel concentrations.	there is accumulating evidence that transenvelope efflux pumps of the resistance, nodulation, cell division protein family (rnd) are excreting toxic substances from the periplasm across the outer membrane directly to the outside. this would mean that resistance of gram-negative bacteria to organic toxins and heavy metals is in fact a two-step process: one set of resistance factors control the concentration of a toxic substance in the periplasm, another one that in the cytoplasm. efficient peripl ...	2005	16158236
requirements for heterologous production of a complex metalloenzyme: the membrane-bound [nife] hydrogenase.	by taking advantage of the tightly clustered genes for the membrane-bound [nife] hydrogenase of ralstonia eutropha h16, broad-host-range recombinant plasmids were constructed carrying the entire membrane-bound hydrogenase (mbh) operon encompassing 21 genes. we demonstrate that the complex mbh biosynthetic apparatus is actively produced in hydrogenase-free hosts yielding fully assembled and functional mbh protein.	2005	16159796
identification and characterization of two polyhydroxyalkanoate biosynthesis loci in pseudomonas sp. strain 3y2.	a pseudomonas strain, 3y2, that produced polyhydroxyalkanoate (pha) polymers consisting of 3-hydroxybutyric acid (3hb) and medium-chain-length 3-hydroxyalkanoate (mcl-ha) units, with up to 30% 3hb, was isolated. two pha biosynthesis loci (pha ( ps-1) and pha ( ps-2)) from 3y2 were cloned by polymerase chain reaction amplification techniques. the pha ( ps-2) locus was similar to the pha biosynthesis loci of other pha-producing pseudomonas strains, with five tandem open reading frames (orfs) locat ...	2005	16175367
cell cycle synchronization of cupriavidus necator by continuous phasing measured via flow cytometry.	the continuous phasing technique was successfully used to obtain a high degree of cell cycle synchrony in cultures of the model organism ralstonia eutropha jmp 134 (today reclassified into cupriavidus necator). the responses of the organism were evaluated with flow cytometric determinations of dna contents and cell size (by fluorescence and forward scatter measurements, respectively, after staining with the dna-binding dye 4',6-diamidino-2'-phenylindole, dapi), and cell concentration, after stai ...	2005	16180241
properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (phazd) in wautersia eutropha h16.	a novel intracellular poly(3-hydroxybutyrate) (phb) depolymerase (phazd) of wautersia eutropha (formerly ralstonia eutropha) h16 which shows similarity with the catalytic domain of the extracellular phb depolymerase in ralstonia pickettii t1 was identified. the positions of the catalytic triad (ser190-asp266-his330) and oxyanion hole (his108) in the amino acid sequence of phazd deduced from the nucleotide sequence roughly accorded with those of the extracellular phb depolymerase of r. pickettii ...	2005	16199568
integrated recombinant protein expression and purification platform based on ralstonia eutropha.	protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. we describe a protein purification scheme wherein ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. this approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydr ...	2005	16204482
organization of polyhydroxyalkanoate synthase for in vitro polymerization as revealed by atomic force microscopy.	individual polyhydroxyalkanoate synthase molecules from ralstonia eutropha (phacre) were directly visualized on highly oriented pyrolytic graphite (hopg) by atomic force microscopy (afm). phacre molecule was observed as a spherical particle of 2.9 +/- 0.4 nm in height and 28 +/- 4 nm in width. in vitro polymerization reaction on hopg was carried out for 5 min by reacting the phacre molecules with (r)-3-hydroxybutyryl-coa monomers. the reaction product was then observed after the removal of water ...	2005	16208629
production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from gluconate and glucose by recombinant aeromonas hydrophila and pseudomonas putida.	aeromonas hydrophila 4ak4 and pseudomonas putida gpp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (phbhhx) using gluconate and glucose rather than fatty acids. a truncated tesa gene, encoding cytosolic thioesterase i of escherichia coli which catalyzes the conversion of acyl-acp into free fatty acids, was introduced into a. hydrophila 4ak4. when grown in gluconate, the recombinant a. hydrophila 4ak4 synthesized 10% (w/w) phbhhx containing 14% (mol/mo ...	2005	16215853
repeated batch cultivation of ralstonia eutropha for poly (beta-hydroxybutyrate) production.	batch cultivation of ralstonia eutropha nrrl b14690 attained 21 g biomass l(-1) and 9.4 g poly(beta-hydroxybutyrate) l(-1) (0.45 g phb g(-1 )dry wt(-1)) in 60 h. repeated batch operation (empty-and-fill protocol) to remove 20% (v/v) of the culture broth and to supplement an equal volume of fresh media resulted in 49 g biomass l(-1) and 25 g phb l(-1) (0.51 g phb g(-1) dry wt(-1)) with an overall productivity of 0.42 g phb l(-1 )h(-1 )in 67 h. in the two cycles of repeated batch fermentation ther ...	2005	16215857
biosynthesis and characterization of poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from palm oil products in a wautersia eutropha mutant.	palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [p(3hb-co-3hhx)] by a mutant strain of wautersia eutropha (formerly ralstonia eutropha) harboring the aeromonas caviae polyhydroxyalkanoate (pha) synthase gene. palm kernel oil was an excellent carbon source for the production of cell biomass and p(3hb-co-3hhx). about 87% (w/w) of the cell dry weight as p(3hb-co-3hhx) was obtained using 5 g palm kernel oil/l ...	2005	16215858
enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with coa recycling using polyhydroxyalkanoate synthase and acyl-coa synthetase.	we succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [p(3 hb-co-4 hb)] synthesis with coa recycling using polyhydroxyalkanoate synthase and an acyl-coa synthetase. using this method, the monomer compositions in p(3 hb-co-4 hb)s could be controlled strictly by the ratios of the monomers in the reaction mixtures.	2005	16233824
a simple solid phase assay for the detection of 2,4-d in soil.	contaminated soils are usually characterized using chemical analyses. however, these do not assess the bioavailability of pollutants, a factor which may be important in estimating the risks associated with contamination. thus there is a need to support chemical analyses with information on biological effects to determine the potential risks a pollutant may pose in the soil. although bacterial bioreporters have been used to detect the presence of contaminants in soils, in general these studies ha ...	2005	16009273
novel intracellular 3-hydroxybutyrate-oligomer hydrolase in wautersia eutropha h16.	wautersia eutropha h16 (formerly ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (phb) with intracellular poly(3-hydroxybutyrate) depolymerases. in this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (phazc) gene was cloned and overexpressed in escherichia coli. then phazc was purified and characterized. immunoblot analysis with polyclonal antiserum against phazc revealed that most phazc is present in the cytosolic fraction and a small amount ...	2005	16030206
amino acids in positions 48, 52, and 73 differentiate the substrate specificities of the highly homologous chlorocatechol 1,2-dioxygenases cbna and tcbc.	chlorocatechol 1,2-dioxygenase (ccd) is the first-step enzyme of the chlorocatechol ortho-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. two ccds, cbna from the 3-chlorobenzoate-degrader ralstonia eutropha nh9 and tcbc from the 1,2,4-trichlorobenzene-degrader pseudomonas sp. strain p51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. but cbna and tcbc are different in substrate ...	2005	16030237
use of a reverse micelle system for study of oligomeric structure of nad+-reducing hydrogenase from ralstonia eutropha h16.	inclusion of an oligomeric enzyme, nad+-dependent hydrogenase from the hydrogen-oxidizing bacterium ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. it was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, hoxhy, is a minimal structural unit catalyzing hydroge ...	2005	16038606
biosynthesis of poly-beta-hydroxyalkanoates by sphingopyxis chilensis s37 and wautersia sp. pzk cultured in cellulose pulp mill effluents containing 2,4,6-trichlorophenol.	poly-beta-hydroxyalkanoates (pha) polymer is synthesized by different bacterial species. there has been considerable interest in the development and production of biodegradable polymers; however, the high cost of pha production has restricted its applications. kraft cellulose industry effluents containing 2,4,6-trichlorophenol (10 or 20 microg ml(-1)) were used by the bacteria sphingopyxis chilensis s37 and wautersia sp. pzk to synthesize pha. in this condition, s. chilensis s37 was able to grow ...	2005	16044293
application of spectrofluorometry to the prediction of phb concentrations in a fed-batch process.	on-line estimation of biopolymer production during fermentation would be a useful adjunct to the development of strategies for process control and optimization. this study examined the applicability of spectrofluorometry, along with other on-line measurements, for the prediction of poly-beta-hydroxybutyric acid (phb) concentrations in a high-cell density fed-batch fermentation of ralstonia eutropha. models previously used for modelling phb evolution with time are not sufficiently accurate for si ...	2005	16047168
site-directed saturation mutagenesis at residue f420 and recombination with another beneficial mutation of ralstonia eutropha polyhydroxyalkanoate synthase.	the f420s substitution enhances the specific activity of ralstonia eutropha pha synthase (phacre). we have now carried out site-directed saturation mutagenesis of f420 of phacre and, amongst the f420 mutants, the f420s mutant gave the highest poly(3-hydroxybutyrate) (phb) content. in vitro activity assay showed that the f420s enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. enhancement of phb accumulation was achieved by combination of the f420s mutati ...	2005	16049738
the structure of the ni-fe site in the isolated hoxc subunit of the hydrogen-sensing hydrogenase from ralstonia eutropha.	the regulatory ni-fe hydrogenase (rh) from ralstonia eutropha which forms a [hoxbc]2 complex functions as a hydrogen sensor under aerobic conditions. we have studied a novel strep-tag isolate of the rh large subunit, hoxc(st), which lacks the fe-s clusters of hoxb, allowing for structure determination of the catalytic site by x-ray absorption spectroscopy both at the ni and, for the first time, also at the fe k-edge. this technique, together with fourier-transform infrared spectroscopy, revealed ...	2005	16051223
internal mass transfer effect on biodegradation of phenol by ca-alginate immobilized ralstonia eutropha.	phenol biodegradation by free and ca-alginate immobilized ralstonia eutropha was performed in batch system. optimum initial ph and temperature were determined as 7 and 30 degrees c, respectively for free cells, while a wide ph and temperature range were obtained for immobilized cells. phenol had a strong inhibitory effect on the microbial growth and haldane model was used to describe the substrate inhibition. model parameters were determined as mumax=0.89 h(-1), ks=55.11 mg dm(-3) and ki=257.94 ...	2005	16051433
hydrogen sensing by enzyme-catalyzed electrochemical detection.	hydrogen (h2) is a possible future alternative to current fossil-based transportation fuels; however, its lower explosive limit in air requires a reliable sensor to detect leaks wherever h2 is produced, stored, or used. most current h2 sensors employ palladium or its alloy as the sensing element, featuring high operating temperature and limited selectivity. in this study, we report using soluble hydrogenase (sh) of aerobic beta-proteobacterium ralstonia eutropha strain h16 to accomplish ambient, ...	2005	16053311
a model system for [nife] hydrogenase maturation studies: purification of an active site-containing hydrogenase large subunit without small subunit.	the large subunit hoxc of the h2-sensing [nife] hydrogenase from ralstonia eutropha was purified without its small subunit. two forms of hoxc were identified. both forms contained iron but only substoichiometric amounts of nickel. one form was a homodimer of hoxc whereas the second also contained the ni-fe site maturation proteins hypc and hypb. despite the presence of the ni-fe active site in some of the proteins, both forms, which lack the fe-s clusters normally present in hydrogenases, cannot ...	2005	16061234
structural and oxidation-state changes at its nonstandard ni-fe site during activation of the nad-reducing hydrogenase from ralstonia eutropha detected by x-ray absorption, epr, and ftir spectroscopy.	structure and oxidation state of the ni-fe cofactor of the nad-reducing soluble hydrogenase (sh) from ralstonia eutropha were studied employing x-ray absorption spectroscopy (xas) at the ni k-edge, epr, and ftir spectroscopy. the sh comprises a nonstandard (cn)ni-fe(cn)(3)(co) site; its hydrogen-cleavage reaction is resistant against inhibition by dioxygen and carbon monoxide. simulations of the xanes and exafs regions of xas spectra revealed that, in the oxidized sh, the ni(ii) is six-coordinat ...	2005	15643882
non-standard structures of the ni-fe cofactor in the regulatory and the nad-reducing hydrogenases from ralstonia eutropha.	spectroscopy on two oxygen-insensitive ni-fe hydrogenases from ralstonia eutropha (nad-reducing, soluble hydrogenase; hydrogen sensor, regulatory hydrogenase) reveals non-standard catalytic behaviour and unique structures of their ni-fe cofactors. possible mechanistic implications are briefly discussed.	2005	15667255
a hydrogen-sensing multiprotein complex controls aerobic hydrogen metabolism in ralstonia eutropha.	h(2) is an attractive energy source for many microorganisms and is mostly consumed before it enters oxic habitats. thus aerobic h(2)-oxidizing organisms receive h(2) only occasionally and in limited amounts. metabolic adaptation requires a robust oxygen-tolerant hydrogenase enzyme system and special regulatory devices that enable the organism to respond rapidly to a changing supply of h(2). the proteobacterium ralstonia eutropha strain h16 that harbours three [nife] hydrogenases perfectly meets ...	2005	15667276
transcriptional regulation of nitric oxide reduction in ralstonia eutropha h16.	nitric oxide reduction in ralstonia eutropha h16 is catalysed by the quinol-dependent no reductase norb. norb and the adjacent nora form an operon that is controlled by the sigma(54)-dependent transcriptional activator norr in response to no. a norr derivative containing male in place of the n-terminal domain binds to a 73 bp region upstream of nora that includes three copies of the putative upstream activator sequence ggt-(n(7))-acc. mutations altering individual bases of this sequence resulted ...	2005	15667304
chlorinated aliphatic hydrocarbon-induced degradation of trichloroethylene in wautersia numadzuensis sp. nov.	two strains of trichloroethylene (tce)-degrading bacteria were isolated from soils at polluted and unpolluted sites. the isolates, strains te26(t) and k6, showed co-substrate-independent tce-degrading activity. tce degradation was accelerated by preincubation with tetrachloroethylene, cis-dichloroethylene (dce) and 1,1-dce. tce-degrading activities of strains te26(t) and k6 were 0.23, 0.24 micromol min(-1) g(-1) dry cells, respectively. 16s rdna sequences of strains te26(t) and k6 were almost id ...	2005	15570416
in vivo monitoring of pha granule formation using gfp-labeled pha synthases.	for the first time a functional protein was fused to a pha synthase resulting in pha granule formation and display of the respective function at the pha granule surface. the gfp reporter protein was n-terminally fused to the class i pha synthase of cupriavidus necator (phac) and the class ii pha synthase of pseudomonas aeruginosa pao1 (phac1), respectively, while maintaining pha synthase activity and pha granule formation. fluorescence microscopy studies of gfp-pha synthase attached to emerging ...	2005	15963662
inducible trans-activation of plastid transgenes: expression of the r. eutropha phb operon in transplastomic tobacco.	deleterious effects of constitutive transgene expression can occur if gene products are harmful to the transformed plant. constraints such as growth inhibition and male sterility have been observed in plastid transformants containing the phb operon encoding the genes required for the production of the polyester polyhydroxybutyric acid (phb). in order to induce phb synthesis in tobacco in a well-timed manner, we have constructed a trans-activation system to regulate transcription of the phb opero ...	2005	15964903
overexpression, purification, crystallization and crystallographic analysis of copk of cupriavidus metallidurans.	copk of cupriavidus metallidurans is a 93-amino-acid protein whose mature form (73 amino acids) has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mm citrate ph 3.5, 200 mm li2so4, 20%(w/v) glycerol, 13%(w/v) peg 8000. crystals display orthorhombic symmetry, with unit-cell parameters a = 57.53, b = 128.65, c = 49.77 a, and diffract to 2.2 a resolution using synchrotron radiation.	2005	16511169
beta-rhizobia from mimosa pigra, a newly discovered invasive plant in taiwan.	a total of 191 rhizobial isolates from the root nodules of three geographically separate populations of the invasive plant mimosa pigra in taiwan were examined using amplified rdna restriction analysis, 16s rdna sequences, protein profiles and elisa. of these, 96% were identified as burkholderia and 4% as cupriavidus taiwanensis. the symbiosis-essential genes noda and nifh were present in two strains of burkholderia (pas44 and ptk47), and in one of c. taiwanensis (pas15). all three could nodulat ...	2005	16313648
proof that burkholderia strains form effective symbioses with legumes: a study of novel mimosa-nodulating strains from south america.	twenty mimosa-nodulating bacterial strains from brazil and venezuela, together with eight reference mimosa-nodulating rhizobial strains and two other beta-rhizobial strains, were examined by amplified rrna gene restriction analysis. they fell into 16 patterns and formed a single cluster together with the known beta-rhizobia, burkholderia caribensis, burkholderia phymatum, and burkholderia tuberum. the 16s rrna gene sequences of 15 of the 20 strains were determined, and all were shown to belong t ...	2005	16269788
electrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels.	use of hydrogen in fuel cells requires catalysts that are tolerant to oxygen and are able to function in the presence of poisons such as carbon monoxide. hydrogen-cycling catalysts are widespread in the bacterial world in the form of hydrogenases, enzymes with unusual active sites composed of iron, or nickel and iron, that are buried within the protein. we have established that the membrane-bound hydrogenase from the beta-proteobacterium ralstonia eutropha h16, when adsorbed at a graphite electr ...	2005	16260746
precipitation of silver-thiosulfate complex and immobilization of silver by cupriavidus metallidurans ch34.	cupriavidus metallidurans ch34 is a facultative chemolithotrophic bacterium that possesses two megaplasmids (pmol28 and pmol30) that confer resistance to eleven metals. the ability of cupriavidus metallidurans ch34 to resist silver is described here. electronic microscopy, energy-dispersive x-ray (edx) and x-ray diffractometry (drx) observations revealed that c. metallidurans ch34 strongly associated silver with the outer membrane, under chloride chemical form. using derivate strains of c. metal ...	2005	16388403
[physiological and biochemical characteristics and capacity for polyhydroxyalkanoates synthesis in a glucose-utilizing strain of hydrogen-oxidizing bacteria, ralstonia eutropha b8562].	the physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain ralstonia eutropha b8562 were investigated in comparison with the parent strain r. eutropha b5786. the morphological, cultural, and biochemical characteristics of strain r. eutropha b8562 were similar to those of strain r. eutropha b5786. genetic analysis revealed differences between the 16s rrna gene sequences of these strains. the growth characteristics of the mutant using glucose as th ...	2005	16400989
[nife]-hydrogenases of ralstonia eutropha h16: modular enzymes for oxygen-tolerant biological hydrogen oxidation.	recent research on hydrogenases has been notably motivated by a desire to utilize these remarkable hydrogen oxidation catalysts in biotechnological applications. progress in the development of such applications is substantially hindered by the oxygen sensitivity of the majority of hydrogenases. this problem tends to inspire the study of organisms such as ralstonia eutropha h16 that produce oxygen-tolerant [nife]-hydrogenases. r. eutropha h16 serves as an excellent model system in that it produce ...	2005	16645314
detection of intermediates from the polymerization reaction catalyzed by a d302a mutant of class iii polyhydroxyalkanoate (pha) synthase.	polyhydroxybutyrate (phb) synthases catalyze the polymerization of (r)-3-hydroxybutyryl-coa (hb-coa) into high molecular weight phb, biodegradable polymers. the class iii phb synthase from allochromatium vinosum is composed of a 1:1 mixture of two approximately 40 kda proteins: phac and phae. previous studies using site-directed mutagenesis and a saturated trimer of hydroxybutyryl-coa have suggested the importance of c149 (in covalent catalysis), h331 (in activation of c149), and d302 (in hydrox ...	2005	15683234
effect of over-expression of phasin gene from aeromonas hydrophila on biosynthesis of copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate.	the gene phapah, encoding the protein phasin that is associated with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (phbhhx) granule of aeromonas hydrophila 4ak4, was cloned and characterized. recombinant strains harboring additional copies of the phasin gene (phapah) and the polyhydroxyalkanoate (pha) synthase gene (phacah) accumulated phbhhx copolyesters consisting of 21 mol% 3-hydroxyhexanoate (3hhx) as compared to 14 mol% 3hhx produced by wild type strain. the molecular weight of phbhhx produ ...	2005	15727816
biosynthesis of polyhydroxyalkanoate (pha) copolymer from fructose using wild-type and laboratory-evolved pha synthases.	eleven laboratory-evolved polyhydroxyalkanoate (pha) synthases which originated from pseudomonas sp. 61-3 enzyme (phac1(ps)), together with the wild-type enzyme, were applied for pha synthesis from fructose using ralstonia eutropha phb(-)4 as a host strain. the evolved phac1(ps) mutants had amino acid substitution(s) at position 325 and/or position 481. in these mutants, serine-325 (s325) was replaced by cysteine (c) or threonine (t), while glutamine-481 (q481) was replaced by lysine (k), methio ...	2005	15729719
influence of homologous phasins (phap) on pha accumulation and regulation of their expression by the transcriptional repressor phar in ralstonia eutropha h16.	phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3hb)] granules and affect their size. recently, three homologues of the phasin protein phap1 were identified in ralstonia eutropha strain h16. the functions of phap2, phap3 and phap4 were examined by analysis of r. eutropha h16 deletion strains (deltaphap1, deltaphap2, deltaphap3, deltaphap4, deltaphap12, deltaphap123 and deltaphap1234). when cells were grown under conditions permissive for poly(3hb) accumulation, t ...	2005	15758228
reduction of unusual iron-sulfur clusters in the h2-sensing regulatory ni-fe hydrogenase from ralstonia eutropha h16.	the regulatory ni-fe hydrogenase (rh) from ralstonia eutropha functions as a hydrogen sensor. the rh consists of the large subunit hoxc housing the ni-fe active site and the small subunit hoxb containing fe-s clusters. the heterolytic cleavage of h(2) at the ni-fe active site leads to the epr-detectable ni-c state of the protein. for the first time, the simultaneous but epr-invisible reduction of fe-s clusters during ni-c state formation was demonstrated by changes in the uv-visible absorption s ...	2005	15764814
characterization and properties of g4x mutants of ralstonia eutropha pha synthase for poly(3-hydroxybutyrate) biosynthesis in escherichia coli.	modification of the type i polyhydroxyalkanoate synthase of ralstonia eutropha (phac(re)) was performed through systematic in vitro evolution in order to obtain improved phac(re) having an enhanced activity of poly(3-hydroxybutyrate) (phb) synthesis in recombinant escherichia coli. for the first time, a beneficial g4d n-terminal mutation important for the enhancement of both phb content in dry cells and phac(re) level in vivo was identified. site-directed saturation mutagenesis at the g4 positio ...	2005	15768438
chloromethylmuconolactones as critical metabolites in the degradation of chloromethylcatechols: recalcitrance of 2-chlorotoluene.	to elucidate possible reasons for the recalcitrance of 2-chlorotoluene, the metabolism of chloromethylcatechols, formed after dioxygenation and dehydrogenation by ralstonia sp. strain ps12 tetrachlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, was monitored using chlorocatechol dioxygenases and chloromuconate cycloisomerases partly purified from ralstonia sp. strain ps12 and wautersia eutropha jmp134. two chloromethylcatechols, 3-chloro-4-methylcatechol and 4-chloro-3-methyl ...	2005	15774876
[the synthesis of hydroxybutyrate and hydroxyvalerate copolymers by the bacterium ralstonia eutropha].	the paper deals with the study of the synthesis of 3-hydroxybutyrate (3hb) and 3-hydroxyvalerate (3hv) copolymers by the bacterium ralstonia eutropha b-5786 grown under different carbon nutrition conditions (growth on carbon dioxide, fructose, and co2-valerate and fructose-valerate mixtures). the parameters to be analyzed included the yield of biomass, the yield, synthesis rate, and composition of copolymers, the activity of the key enzymes of polyhydroxyalkanoate (pha) synthesis (beta-ketothiol ...	2005	15835780
the soluble nad+-reducing [nife]-hydrogenase from ralstonia eutropha h16 consists of six subunits and can be specifically activated by nadph.	the soluble [nife]-hydrogenase (sh) of the facultative lithoautotrophic proteobacterium ralstonia eutropha h16 has up to now been described as a heterotetrameric enzyme. the purified protein consists of two functionally distinct heterodimeric moieties. the hoxhy dimer represents the hydrogenase module, and the hoxfu dimer constitutes an nadh-dehydrogenase. in the bimodular form, the sh mediates reduction of nad(+) at the expense of h(2). we have purified a new high-molecular-weight form of the s ...	2005	15838039
oxygen tolerance of the h2-sensing [nife] hydrogenase from ralstonia eutropha h16 is based on limited access of oxygen to the active site.	hydrogenases, abundant proteins in the microbial world, catalyze cleavage of h2 into protons and electrons or the evolution of h2 by proton reduction. hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. we have selected the h2-sensing regulatory [nife] hydrogenase of ralstonia eutropha h16 to investiga ...	2005	15849358
kinetic studies of polyhydroxybutyrate granule formation in wautersia eutropha h16 by transmission electron microscopy.	wautersia eutropha, formerly known as ralstonia eutropha, a gram-negative bacterium, accumulates polyhydroxybutyrate (phb) as insoluble granules inside the cell when nutrients other than carbon are limited. in this paper, we report findings from kinetic studies of granule formation and degradation in w. eutropha h16 obtained using transmission electron microscopy (tem). in nitrogen-limited growth medium, the phenotype of the cells at the early stages of granule formation was revealed for the fir ...	2005	15901706
analysis of transient polyhydroxybutyrate production in wautersia eutropha h16 by quantitative western analysis and transmission electron microscopy.	polyhydroxybutyrates (phbs) are polyoxoesters generated from (r)3-hydroxybutyryl coenzyme a by phb synthase. during the polymerization reaction, the polymers undergo a phase transition and generate granules. wautersia eutropha can transiently accumulate phb when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [tsb]). phb homeostasis under these growth conditions was examined by quantitative western analysis to monitor the proteins presen ...	2005	15901707
transcriptional analysis of ralstonia eutropha genes related to poly-(r)-3-hydroxybutyrate homeostasis during batch fermentation.	poly-(r)-3-hydroxybutyrate (phb) homeostasis in ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic phb granule. phb synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. reverse transcriptase quantitative pcr and quantitative western blotting were carried out on batch cultures of ...	2005	15924243
a simple structured mathematical model for biopolymer (phb) production.	economic production technology for a biodegradable polymer (poly-beta-hydroxybutyrate, phb) is urgently required to replace conventional polymers, which have an inherent disadvantage of staying in the environment forever. various approaches have been applied for improving the productivity and reducing the production cost, which are considered to be the two major problems associated with industrial production of phb. one of the engineering approaches to improve phb productivity could be to design ...	2005	15932263
class iii polyhydroxybutyrate synthase: involvement in chain termination and reinitiation.	polyhydroxybutyrate (phb) synthase catalyzes the polymerization of (r)-3-hydroxybutyryl-coa (coa = coenzyme a) into high molecular weight phb. recombinant wild-type (wt) class iii synthase from allochromatium vinosum (phacphae(av)), antibodies to this synthase and to phb, and [(14)c]hydroxybutyryl-coa (hb-coa) have been used to detect oligomeric hydroxybutyrate (hb) units covalently bound to the synthase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. although a distrib ...	2005	15938626
polyhydroxyalkanoate (pha) granule formation in ralstonia eutropha cells: a computer simulation.	computer simulation of polyhydroxyalkanoate (pha) granule formation in vivo could help to design strategies to optimize the fermentation process and achieve higher yields of pha. it could also suggest biotechnological approaches to control the granule size and molecular weight of the polymer. a computer program simulating the formation of pha granules inside a ralstonia eutropha cell was developed, based on published experimental data. the results are applicable to r. eutropha cells or other mic ...	2004	14758517
sequential feeding of glucose and valerate in a fed-batch culture of ralstonia eutropha for production of poly(hydroxybutyrate-co-hydroxyvalerate) with high 3-hydroxyvalerate fraction.	several important properties of poly(3-hydroxybutyric-co-3-hydroxyvaleric acids) (p(3hb-co-3hv) depend mainly on the hv unit fraction of the copolymer. sequential and simultaneous feeding of glucose and valerate were employed to produce p(3hb-co-3hv) in a fed-batch culture of ralstonia eutropha, and the effects of feeding models on the cell growth, 3hv unit fraction, and copolymer productivity have been investigated. the sequential feeding of glucose and then valerate resulted in a cell density ...	2004	14763836
selective release and function of one of the two fmn groups in the cytoplasmic nad+-reducing [nife]-hydrogenase from ralstonia eutropha.	the soluble, cytoplasmic nad+-reducing [nife]-hydrogenase from ralstonia eutropha is a heterotetrameric enzyme (hoxfuyh) and contains two fmn groups. the purified oxidized enzyme is inactive in the h2-nad+ reaction, but can be activated by catalytic amounts of nadh. it was discovered that one of the fmn groups (fmn-a) is selectively released upon prolonged reduction of the enzyme with nadh. during this process, the enzyme maintained its tetrameric form, with one fmn group (fmn-b) firmly bound, b ...	2004	14764097
characterization of a gene cluster encoding the maleylacetate reductase from ralstonia eutropha 335t, an enzyme recruited for growth with 4-fluorobenzoate.	a gene cluster containing a gene for maleylacetate reductase (ec 1.3.1.32) was cloned from ralstonia eutropha 335(t) (dsm 531(t)), which is able to utilize 4-fluorobenzoate as sole carbon source. sequencing of this gene cluster showed that the r. eutropha 335(t) maleylacetate reductase gene, maca, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. it otherwise comprises a gene for a hypothetical membrane transport protein, macb, pos ...	2004	14766925
the h2-sensing complex of ralstonia eutropha: interaction between a regulatory [nife] hydrogenase and a histidine protein kinase.	two [nife] hydrogenases enable the proteobacterium ralstonia eutropha h16 to grow on molecular hydrogen as the sole energy source. a third [nife] hydrogenase (rh) acts as an h2 sensor in a multiple component signal transduction chain that controls hydrogenase gene transcription. the rh forms a dimeric heterodimer (hoxbc)2 in which hoxc contains the h2-sensing active site and hoxb the electron-transferring components including an organic, not yet identified redox cofactor. this oligomer forms a t ...	2004	15009894
wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including ralstonia eutropha and related species, and proposal of ralstonia [pseudomonas] syzygii (roberts et al. 1990) comb. nov.	comparative 16s rdna sequence analysis indicates that two distinct sublineages, with a sequence dissimilarity of >4 % (bootstrap value, 100 %), exist within the genus ralstonia: the ralstonia eutropha lineage, which comprises ralstonia basilensis, ralstonia campinensis, r. eutropha, ralstonia gilardii, ralstonia metallidurans, ralstonia oxalatica, ralstonia paucula, ralstonia respiraculi and ralstonia taiwanensis; and the ralstonia pickettii lineage, which comprises ralstonia insidiosa, ralstoni ...	2004	15023939
reliable, sensitive, rapid and quantitative enzyme-based assay for gamma-hydroxybutyric acid (ghb).	several assays for gamma-hydroxybutyrate (4-hydroxybutyrate, ghb) have been developed based on the enzyme gamma-hydroxybutyrate dehydrogenase (ghb-dh). enzymatic oxidation of ghb by nad+ is coupled to diaphorase-mediated reduction of pro-dye to yield colored product. ghb-dh from ralstonia eutropha was cloned and expressed as a stable fusion protein easily purified by affinity chromatography. quantitative initial velocity and endpoint versions of the assay in solution are described. michaelis-men ...	2004	15027565
biosynthesis of r-3-hydroxyalkanoic acids by metabolically engineered escherichia coli.	an efficient system for the production of (r)-hydroxyalkanoic acids (rhas) was developed in natural polyhydroxyalkanoate (pha)-producing bacteria and recombinant escherichia coli. acidic alcoholysis of purified pha and in vivo depolymerization of pha accumulated in the cells allowed the production of rhas. in recombinant e. coli, rha production was achieved by removing coa from (r)-3-hydroxyacyl-coa and by in vivo depolymerization of pha. when the recombinant e. coli harboring the ralstonia eutr ...	2004	15054264
[dynamics of activity of the key enzymes of polyhydroxyalkanoate metabolism in ralstonia eutropha].	the dynamics of accumulation of polyhydroxybutyrate (phb) and the activities of the key enzymes of phb metabolism (beta-ketothiolase, acetoacetyl-coa reductase, pha synthase, d-hydroxybutyrate dehydrogenase, and pha depolymerase) in the hydrogen bacterium ralstonia eutropha b5786 were studied under various conditions of carbon nutrition and substrate availability. the highest activities of beta-ketothiolase, acetoacetyl-coa reductase, and pha synthase were recorded at the stage of acceleration o ...	2004	15125198
enzymatic surface-initiated polymerization: a novel approach for the in situ solid-phase synthesis of biocompatible polymer poly(3-hydroxybutyrate).	a novel system for surface-initiated enzymatic polymerization of a film of polyhydroxyalkanoate (pha) on solid surfaces has been developed and characterized. phas are aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy, and their properties range from elastomers to thermoplastics, depending on their monomeric composition. the pha synthase from ralstonia eutropha h16 was expressed as a poly-histidine fusion in escherichia coli and immobilized onto severa ...	2004	15132678
modal difference in comonomer-unit compositional distributions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)s biosynthesized by two strains, ralstonia eutropha and alcaligenes latus.		2004	15132709
the soluble [nife]-hydrogenase from ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen.	infrared spectra of (15)n-enriched preparations of the soluble cytoplasmic nad(+)-reducing [nife]-hydrogenase from ralstonia eutropha are presented. these spectra, together with chemical analyses, show that the ni-fe active site contains four cyanide groups and one carbon monoxide molecule. it is proposed that the active site is a (rs)(2)(cn)ni(micro-rs)(2)fe(cn)(3)(co) centre (r=cys) and that h(2) activation solely takes place on nickel. one of the two fmn groups (fmn-a) in the enzyme can be re ...	2004	15164270
adaptive responses to static conditions in nutrient-rich cultures of luminous ralstonia eutropha.	the lux-gene fused ralstonia eutropha, when adapting to static conditions, causes stratification of air-exposed and nutrient-rich cultures at above 0.15 mg biomass ml(-1). the o2 respiring biofilm (luminous neuston) phase, along with the dark sub-neustonic suspension phase, develops within 5-60 min. the instability of the biphasic static culture was identified as a reason for occasionally observable oscillatory bioluminescence.	2004	15168854
roles of poly(3-hydroxybutyrate) depolymerase and 3hb-oligomer hydrolase in bacterial phb metabolism.	many poly-3-hydroxybutyrate (phb)-degrading enzymes have been studied. but biological roles of 3hb-oligomer hydrolases (3hbohs) and how phb depolymerases (phbdps) and 3hbohs cooperate in phb metabolism are not fully elucidated. in this study, several phbdps and 3hbohs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. from the results, phbdp and 3hboh seemed to play a role in phb metabolism in three types o ...	2004	15170237
genetic organization of the catabolic plasmid pjp4 from ralstonia eutropha jmp134 (pjp4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.	ralstonia eutropha jmp134 (pjp4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, incp-1 beta plasmid pjp4. the complete sequence of the 87,688 nucleotides of pjp4, encoding 83 open reading frames (orfs), is reported. most of the coding sequence corresponds to a well-conserved incp-1 beta backbone and the previously reported tfd genes. in addition, w ...	2004	15186344
transcription level of granule-associated phap and phar genes and granular morphogenesis of poly-beta-hydroxyalkanoate granules in ralstonia eutropha.	the transcription levels of the granule-associated phap and phar genes in ralstonia eutropha were regulated through the transformation of the phbc genes from r. eutropha and alcaligenes latus into the poly-beta-hydroxybutyrate synthase-negative mutant. the granular morphogenesis of short chain length, poly-beta-hydroxyalkanoate (scl-pha) was closely associated with the mrna transcription levels of the phap and phar genes, especially with the ratio of phap/phar genes. the phasin protein encoded b ...	2004	15200169
chlorophenol removal from soil suspensions: effects of a specialised microbial inoculum and a degradable analogue.	two soils of different contamination history were tested in slurry for their self-remediability towards mono-, di- and trisubstituted chlorophenols. the landfill soil showed poor ability in removing the compounds. instead, the soil from the golf course, treated for many years with a 2,4,6-trichlorophenol derivative (prochloraz), remediated different concentrations of the same 2,4,6tcp, 2,4-dichlorophenol and monochlorophenol isomers, singly and in mixtures, at varying degradation rates. ralstoni ...	2004	15228073
effective enhancement of short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production by coexpression of genetically engineered 3-ketoacyl-acyl-carrier-protein synthase iii (fabh) and polyhydroxyalkanoate synthesis genes.	polyhydroxyalkanoates (phas) are biodegradable polyesters that have a wide variety of physical properties dependent on the lengths of the pendant groups of the monomer units in the polymer. phas composed of mostly short-chain-length (scl) monomers are often stiff and brittle, whereas phas composed of mostly medium-chain-length (mcl) monomers are elastomeric in nature. scl-mcl pha copolymers can have properties between the two states, dependent on the ratio of scl and mcl monomers in the copolyme ...	2004	15244465
identification of the anabaena sp. strain pcc7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like pseudomonas putida and ralstonia eutropha.	the cyanophycin synthetase gene cpha1 encoding the major cyanophycin synthetase (cpha) of anabaena sp. strain pcc7120 was expressed in escherichia coli conferring so far the highest specific cpha activity to e. coli (6.7 nmol arginine per min and mg protein). cpha1 and cpha genes of synechocystis sp. strains pcc6803 and pcc6308 and synechococcus strain ma19 were also expressed in wild types and polyhydroxyalkanoate-negative (pha) mutants of pseudomonas putida and ralstonia eutropha. recombinant ...	2004	15244482
mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from ralstonia eutropha.	molybdenum enzymes containing the pterin cofactor are a diverse group of enzymes that catalyse in general oxygen atom transfer reactions. aiming at studying the amino acid residues, which are important for the enzymatic specificity, we used nitrate reductase from ralstonia eutropha (r.e.nap) as a model system for mutational studies at the active site. we mutated amino acids at the mo active site (cys181 and arg421) as well as amino acids in the funnel leading to it (met182, asp196, glu197, and t ...	2004	15249219
a hydrogen-evolving enzyme is present in frankia sp. r43.	the ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes frankia sp. r43 and frankia sp. kb5. to further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) frankia sp. r43 but not frankia sp. kb5 evolved hydrogen,which was not linked to nad-reducing activity. immunological analysis o ...	2004	15251202
the complex structure of polyhydroxybutyrate (phb) granules: four orthologous and paralogous phasins occur in ralstonia eutropha.	analysis of the genome sequence of the polyhydroxyalkanoate- (pha) accumulating bacterium ralstonia eutropha strain h16 revealed three homologues (phap2, phap3 and phap4) of the phasin protein phap1. phap1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3hb), granules. phap2, phap3 and phap4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to phap1, respectively. the calculated molecular masses of phap1, phap2, phap3 and pha ...	2004	15256572
[study of ralstonia eutropha culture producing polyhydroxyalkanoates on products of coal processing].	kinetic indices of growth, polyhydroxyalkanoate (pha) accumulation, and gas exchange have been studied in a culture of the carbon monoxide-resistant hydrogen strain ralstonia eutropha b-5786 grown on a gaseous substrate (gs) obtained by lignite gasification. the gs was shown to be suitable for pha production. to increase the degree of gs consumption, various modes of gas supply to the culture were tested. based on the results, an algorithm was developed for calculating and controlling gas-exchan ...	2004	15283331
metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in ralstonia eutropha on short chain fatty acids.	short chain fatty acids such as acetic, propionic, and butyric acids can be synthesized into polyhydroxyalkanoates (phas) by ralstonia eutropha. metabolic carbon fluxes of the acids in living cells have significant effect on the yield, composition, and thermomechanical properties of pha bioplastics. based on the general knowledge of central metabolism pathways and the unusual metabolic pathways in r. eutropha, a metabolic network of 41 bioreactions is constructed to analyze the carbon fluxes on ...	2004	15296425
escherichia coli hmp, an "oxygen-binding flavohaemoprotein", produces superoxide anion and self-destructs.	escherichia coli hmp is a homologue of ralstonia eutropha fhp, the first reported bacterial flavohaemoglobin, and functions in no detoxification. photolysis of co-ligated hmp in the presence of oxygen gave a photodissociable oxy species with k(on) 2.82x10(7) m(-1) s(-1) and k(off) 4.49x10(3) s(-1). the dissociation constant of the primary o(2) compound was 160 microm (25 degrees c, ph 7.0). in order to detect superoxide formation, ferric horseradish peroxidase was used. hmp formed the oxy compou ...	2004	15340787
the role of the active site-coordinating cysteine residues in the maturation of the h2-sensing [nife] hydrogenase from ralstonia eutropha h16.	the h(2)-splitting active site of [nife] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. in this study, the nickel-binding cysteine residues of hoxc, the large subunit of the h(2)-sensing regulatory hydrogenase (rh) from ralstonia eutropha, were replaced by serine. all four mutant proteins, c60s, c63s, c479s, and c482s, were inactive both in h(2) sensing and h(2) oxidation and did not adopt the native oligomeric structure of the rh. nickel was bound only ...	2004	15340794
the auxiliary protein hypx provides oxygen tolerance to the soluble [nife]-hydrogenase of ralstonia eutropha h16 by way of a cyanide ligand to nickel.	the hypx gene of the facultative lithoautotrophic bacterium ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the [nife]-hydrogenases in the bacterium. a deletion of the hypx gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble nad+ -reducing hydrogenase. the enzymatic and infrared spectral properties of the sol ...	2004	15342627
increasing the carbon flux toward synthesis of short-chain-length--medium-chain-length polyhydroxyalkanoate in the peroxisome of saccharomyces cerevisiae through modification of the beta-oxidation cycle.	short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in saccharomyces cerevisiae from intermediates of the beta-oxidation cycle by expressing the polyhydroxyalkanoate synthases from aeromonas caviae and ralstonia eutropha in the peroxisomes. the quantity of polymer produced was increased by using a mutant of the beta-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward r-3-hydroxybutyryl coenzyme a.	2004	15345460
genetic analysis of phenoxyalkanoic acid degradation in sphingomonas herbicidovorans mh.	phenoxyalkanoic acid degradation is well studied in beta- and gammaproteobacteria, but the genetic background has not been elucidated so far in alphaproteobacteria. we report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium sphingomonas herbicidovorans mh and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-d). two genes for alpha-ketoglutarate-dependent dioxygenases, sd ...	2004	15466552
microbial polythioesters.	this feature article describes the current knowledge on biosynthesis of polythioesters (ptes), which are exclusively accumulated by microorganisms capable to synthesize the well-known polyhydroxyalkanoates (phas). two bacterial pte biosynthesis systems are discussed, both are depending on the cultivation conditions and appropriate feeding regimes. the first system comprises the production of pte copolymers by ralstonia eutropha, and the second system has been established in recombinant escherich ...	2004	15468206
biosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes.	a new strategy for bacterial polyhydroxyalkanoate (pha) production by recombinant ralstonia eutropha phb(-)4 harboring mutated pha synthase genes (phac(ac)) from aeromona caviae was investigated. the strain harboring wild-type phac(ac) gene produced a pha copolymer consisting of (r)-3-hydroxybutyrate and (r)-3-hydroxyhexanoate [p(3hb-co-3hhx)] with 3.5 mol-% of 3hhx fraction from soybean oil. when the mutants of phac(ac) gene were applied to this production system, 3hhx fraction in copolymers wa ...	2004	15468213
mass spectrometry feedback control for synthesis of polyhydroxyalkanoate granule microstructures in ralstonia eutropha.	polyhydroxyalkanoate (pha) granules with core-shell layered microstructure were synthesized in ralstonia eutropha using periodic feeding of valeric acid into a growth medium containing excess fructose. the o2 consumption and co2 evolution rates, determined by off-gas mass spectrometry, have been used as sensitive measures to indicate the type of nutrients utilized by r. eutropha during pha synthesis. domains of poly-3-hydroxybutyrate (phb) were formed during polymer storage conditions when only ...	2004	15468214
metabolic engineering for the production of copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by aeromonas hydrophila.	aeromonas hydrophila 4ak4 was able to synthesize copolyesters consisting of 3-hydroxybutyrate (3hb) and about 15 mol-% 3-hydroxyhexanoate (3hhx) (phbhhx) when grown in long chain fatty acids such as dodecanoate regardless of growth conditions. to regulate the unit fraction in phbhhx, phba and phbb genes encoding beta-ketothiolase and acetoacetyl-coa reductase in ralstonia eutropha, were introduced into a. hydrophila 4ak4. when gluconate was used as cosubstrate of dodecanoate, the recombinant pro ...	2004	15468215
the "intracellular" poly(3-hydroxybutyrate) (phb) depolymerase of rhodospirillum rubrum is a periplasm-located protein with specificity for native phb and with structural similarity to extracellular phb depolymerases.	rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (phb) depolymerase system consisting of a soluble phb depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (j. m. merrick and m. doudoroff, j. bacteriol. 88:60-71, 1964). in this study we reinvestigated the soluble r. rubrum phb depolymerase (phaz1). it turned out that phaz1 is a novel type of phb depolymerase with unique properties. purified phaz1 was specific for amorphous short-chain-len ...	2004	15489436
nife hydrogenase active site biosynthesis: identification of hyp protein complexes in ralstonia eutropha.	biosynthesis of the nife hydrogenase active site is a complex process involving the action of the hyp proteins: hypa-hypf. here we investigate the mechanism of nife site biosynthesis in ralstonia eutropha by examining the interactions between hypc, hypd, hype, and hypf1. using an affinity purification procedure based on the strep-tag ii, we purified hypc and hype from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological ana ...	2004	15491154
mutation effects of a conserved alanine (ala510) in type i polyhydroxyalkanoate synthase from ralstonia eutropha on polyester biosynthesis.	type i polyhydroxyalkanoate (pha) synthases, as represented by ralstonia eutropha enzyme (phac(re)), have narrow substrate specificity toward (r)-3-hydroxyacyl-coenzyme a with acyl chain length of c3-c5 to yield pha polyesters. in this study, saturation point mutagenesis of a highly conserved alanine at position 510 (a510) in phac(re) was carried out to investigate the effects on the polymerization activity and the substrate specificity for in vivo pha biosynthesis in bacterial cells. a series o ...	2004	15508175
poly(3-hydroxybutyrate) biosynthesis in the biofilm of alcaligenes eutrophus, using glucose enzymatically released from pulp fiber sludge.	glucose, enzymatically released from pulp fiber sludge, was combined with inorganic salts and used as a growth medium for alcaligenes eutrophus, a gram-negative strain producing poly(3-hydroxybutyrate) (phb). by controlling the concentrations of the inorganic salts in the growth medium, almost 78% of the cell mass was converted to pure phb. efforts were made to find conditions for bacterial growth in the form of a biofilm on a cheap and reusable carrier. a number of positively charged carriers w ...	2004	15528544
engineering of chimeric class ii polyhydroxyalkanoate synthases.	pha synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (phas). using a combinatorial genetic strategy to create unique chimeric class ii pha synthases, we have obtained a number of novel chimeras which display improved catalytic properties. to engineer the chimeric pha synthases, we constructed a synthetic phac gene from pseudomonas oleovorans (phac1po) that was devoid of an internal 540-bp fragment. randomly amplified pcr products (created with primers based on conse ...	2004	15528546
taxonomy of the genus cupriavidus: a tale of lost and found.	dna-dna hybridization experiments and an evaluation of phenotypic characteristics, dna base ratios and 16s rrna gene sequences demonstrated that wautersia eutropha (davies 1969) vaneechoutte et al. 2004, the type species of the genus wautersia, is a later synonym of cupriavidus necator makkar and casida 1987, the type species of the genus cupriavidus. in conformity with rules 15, 17, 23a and 37a(1) of the international code of nomenclature of bacteria, the genus name cupriavidus has priority ove ...	2004	15545472
the chromosomally encoded cation diffusion facilitator proteins dmef and fief from wautersia metallidurans ch34 are transporters of broad metal specificity.	genomic sequencing of the beta-proteobacterium wautersia (previously ralstonia) metallidurans ch34 revealed the presence of three genes encoding proteins of the cation diffusion facilitator (cdf) family. one, czcd, was previously found to be part of the high-level metal resistance system czc that mediates the efflux of co(ii), zn(ii), and cd(ii) ions catalyzed by the czccba cation-proton antiporter. the second cdf protein, fief, is probably mainly a ferrous iron detoxifying protein but also medi ...	2004	15547276
high level recombinant protein expression in ralstonia eutropha using t7 rna polymerase based amplification.	we report further development of a novel recombinant protein expression system based on the gram-negative bacterium, ralstonia eutropha. in this study, we were able to express soluble, active, organophosphohydrolase (oph), a protein that is prone to inclusion body formation in escherichia coli, at titers greater than 10 g/l in high cell density fermentation. this represents a titer that is approximately 100-fold greater than titers previously reported in e. coli for this enzyme. r. eutropha stra ...	2004	15555942
effect of process variables on supercritical fluid disruption of ralstonia eutropha cells for poly(r-hydroxybutyrate) recovery.	this research focuses on the disruption of the gram-negative bacterium ralstonia eutropha cells by supercritical co2 for poly(r-hydroxybutyrate) (phb) recovery. the variables affecting cell disruption such as drying strategy, type of modifier, and cultivation time, as well as operating pressure, temperature, and repeated release of supercritical co2 pressure, have been studied. effect of this disruption technique on phb molecular mass was also investigated. phb recovery was examined using a comb ...	2004	15575709
[physicochemical properties of two-component polyhydroxyalkanoates based on 3-hydroxybutyrate and 3-hydroxyvalerate].	a series of two-component polyhydroxyalkanoates consisting of hydroxybutyrate and hydroxyvalerate monomer at different ratios were synthesized using the bacterium ralstonia eutropha b5786. the properties of polyhydroxyalkanoates were compared with those of the homopolymer of hydroxybutyric acid by x-ray structure analysis, ir spectroscopy, differential scanning calorimetry, and viscosimetry. with an increase in the molar fraction of hydroxyvalerate, an equalization of the ratio of the crystallin ...	2004	15612544
production of poly(3-hydroxybutyrate) by solid-state fermentation with ralstonia eutropha.	the use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (phas) by ralstonia eutropha. two agroindustrial residues (babassu and soy cake) were evaluated as culture media. the maximum poly(hydroxybutyrate) (phb) yield was 1.2 mg g(-1) medium on soy cake in 36 h, and 0.7 mg g(-1) medium on babassu cake in 84 h. addition of 2.5% (w/w) sugar cane molasses to soy cake increased phb production to 4.9 mg g(-1) medium in 60 h. under these con ...	2004	15672227
non-conventional yeasts as producers of polyhydroxyalkanoates--genetic engineering of arxula adeninivorans.	the non-conventional yeast arxula adeninivorans was equipped with the genes phba, phbb and phbc of the polyhydroxyalkanoate (pha) biosynthetic pathway of ralstonia eutropha, which encode beta-ketothiolase, nadph-linked acetoacetyl-coa reductase and pha synthase, respectively. arxula strains transformed solely with the pha synthase gene (phbc) were able to produce pha. however, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (phb) and 0.112% pol ...	2004	14655026
a monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions.	ralstonia eutropha jmp134 2,4,6-trichlorophenol (2,4,6-tcp) 4-monooxygenase catalyzes sequential dechlorinations of 2,4,6-tcp to 6-chlorohydroxyquinol. although 2,6-dichlorohydroxyquinol is a logical metabolic intermediate, the enzyme hardly uses it as a substrate, implying it may not be a true intermediate. evidence is provided to support the proposition that the monooxygenase oxidized 2,4,6-tcp to 2,6-dichloroquinone that remained with the enzyme and got hydrolyzed to 2-chlorohydroxyquinone, w ...	2004	14662756