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genetic analysis of comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer.the polyhydroxyalkanoate (pha) synthase gene of comamonas acidovorans ds-17 (phacca) was cloned by using the synthase gene of alcaligenes eutrophus as a heterologous hybridization probe. complete sequencing of a 4.0-kbp smai-hindiii (sh40) subfragment revealed the presence of a 1,893-bp pha synthase coding region which was followed by a 1,182-bp beta-ketothiolase gene (phaaca). both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary s ...19989726894
pseudomonas sp. strain 273, an aerobic alpha, omega-dichloroalkanedegrading bacterium.a gram-negative, aerobic bacterium was isolated from soil; this bacterium grew in 50% (vol/vol) suspensions of 1,10-dichlorodecane (1,10-dcd) as the sole source of carbon and energy. phenotypic and small-subunit ribosomal rna characterizations identified the organism, designated strain 273, as a member of the genus pseudomonas. after induction with 1,10-dcd, pseudomonas sp. strain 273 released stoichiometric amounts of chloride from c5 to c12 alpha, omega-dichloroalkanes in the presence of oxyge ...19989726906
a new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. the phag gene from pseudomonas putida kt2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme a transferase.to investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (pha) synthesis, we isolated mutants of pseudomonas putida kt2440 deficient in this metabolic route. the gene phag was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhla gene product, which is involved in the rhamnolipid ...19989727022
the genes ruba and rubb for alkane degradation in acinetobacter sp. strain adp1 are in an operon with estb, encoding an esterase, and oxyr.alkanes are oxidized in acinetobacter sp. strain adp1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase. ruba and rubb encode rubredoxin and a nad(p)h-dependent rubredoxin reductase. we demonstrate here that single base pair substitutions in ruba or rubb lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. differences in the degradation capacity for hexadecane and dodecane in these mutan ...199910400587
carbon-source-dependent expression of the palkb promoter from the pseudomonas oleovorans alkane degradation pathway.pseudomonas oleovorans gpo1 can metabolize medium-chain-length alkanes by means of an enzymatic system whose induction is regulated by the alks protein. in the presence of alkanes, alks activates the expression of promoter palkb, from which most of the genes of the pathway are transcribed. in addition, expression of the first enzyme of the pathway, alkane hydroxylase, is known to be influenced by the carbon source present in the growth medium, indicating the existence of an additional overimpose ...19989748457
biocatalyst engineering by assembly of fatty acid transport and oxidation activities for in vivo application of cytochrome p-450bm-3 monooxygenase.the application of whole cells containing cytochrome p-450bm-3 monooxygenase [ec 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to omega-1, omega-2, and omega-3 hydroxy fatty acids was investigated. we utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. for this purpose, escherichia coli recombinants containing plasmid pcyp102 producing the fatty acid monooxygenase cytochrome p-450bm-3 wer ...19989758800
involvement of the cis/trans isomerase cti in solvent resistance of pseudomonas putida dot-t1e.pseudomonas putida dot-t1e is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. we have cloned and sequenced the cti gene of this strain, which encodes the cis/trans isomerase, termed cti, that catalyzes the cis-trans isomerization of esterified fatty acids in phospholipids, mainly cis-oleic acid (c(16:1,9)) and cis-vaccenic acid (c(18:1,11)), in response to solvents. to determine the importance of this cis/trans isomerase for solvent resistance a ...199910482510
diversity in butane monooxygenases among butane-grown bacteria.butane monooxygenases of butane-grown pseudomonas butanovora, mycobacterium vaccae job5, and an environmental isolate, cf8, were compared at the physiological level. the presence of butane monooxygenases in these bacteria was indicated by the following results. (i) o(2) was required for butane degradation. (ii) 1-butanol was produced during butane degradation. (iii) acetylene inhibited both butane oxidation and 1-butanol production. the responses to the known monooxygenase inactivator, ethylene, ...199910508093
metabolic engineering of poly(3-hydroxyalkanoates): from dna to plastic.poly(3-hydroxyalkanoates) (phas) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. a wealth of biological diversity in pha formation exists, with at least 100 different pha constituents and at least five different dedicated pha biosynthetic pathways. this diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic eng ...199910066830
engineering of a stable whole-cell biocatalyst capable of (s)-styrene oxide formation for continuous two-liquid-phase applications.recombinant strains of pseudomonas putida kt2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based escherichia coli recombinants. such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking- ...199910584030
synthesis of alkane hydroxylase of pseudomonas oleovorans increases the iron requirement of alk+ bacterial strains.the alk genes enable pseudomonas oleovorans to utilize alkanes as sole carbon and energy source. expression of the alk genes in p. oleovorans and in two escherichia coli recombinants induced iron limitation in minimal medium cultures. further investigation showed that the expression of the alkb gene, encoding the integral cytoplasmic membrane protein alkb, was responsible for the increase of the iron requirement of e. coli w3110 (pgec47). alkb is the non-heme iron monooxygenase component of the ...199810099198
medium chain length alkane solvent-cell transfer rates in two-liquid phase, pseudomonas oleovorans culturesthe oxidation of medium chain length alkanes and alkenes (c6 to c12) by pseudomonas oleovorans and related, biocatalytically active recombinant organisms, in two-liquid phase cultures can be used for the biochemical production of several interesting fine chemicals. the volumetric productivities that can be attained in two-liquid phase systems can be, in contrast to aqueous fermentations, limited by the transport of substrates from an apolar phase to the cells residing in the aqueous phase and by ...199810099401
a set of genes encoding a second toluene efflux system in pseudomonas putida dot-t1e is linked to the tod genes for toluene metabolism.sequence analysis in pseudomonas putida dot-t1e revealed a second toluene efflux system for toluene metabolism encoded by the ttgdef genes, which are adjacent to the tod genes. the ttgdef genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. to characterize the contribution of the ttgdef system to toluene tolerance in p. putida, site-directed mutagenesis was used to knock out the gene in the wild-type dot-t1e strain and in a ...200010648517
reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in pseudomonas putida.poly(3-hydroxyalkanoates) (phas) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. industrial processing of pha involves purification of the pha granules from high-cell-density cultures. after the fermentation process, cel ...199910103246
nahw, a novel, inducible salicylate hydroxylase involved in mineralization of naphthalene by pseudomonas stutzeri an10.two genes, nahg and nahw, encoding two independent salicylate 1-hydroxylases have been identified in the naphthalene-degrading strain pseudomonas stutzeri an10. while nahg resides in the same transcriptional unit as the meta-cleavage pathway genes, forming the naphthalene degradation lower pathway, nahw is situated outside but in close proximity to this transcriptional unit. the nahg and nahw genes of p. stutzeri an10 are induced and expressed upon incubation with salicylate, and the enzymes tha ...199910197990
microbial desulfurization of a crude oil middle-distillate fraction: analysis of the extent of sulfur removal and the effect of removal on remaining sulfur.rhodococcus sp. strain ecrd-1 was evaluated for its ability to desulfurize a 232 to 343 degrees c middle-distillate (diesel range) fraction of oregon basin (ob) crude oil. ob oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined. gas chromatography (gc), flame ionization detection, and gc sulfur chemiluminesce detection analysis were used to qualitatively evaluate ...19999872778
polyhydroxyalkanoate inclusion body-associated proteins and coding region in bacillus megaterium.polyhydroxyalkanoic acids (pha) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. this work presents the results of a study of pha inclusion body-associated proteins and an analysis of their coding region in bacillus megaterium 11561. a 7, 917-bp fragment of dna was cloned and shown to carry a 4,104-bp cluster of 5 pha genes, phap, -q, -r, -b, and -c. the phap and -q genes were shown to be transcribed ...19999882674
a novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium pseudomonas abietaniphila bkme-9.pseudomonas abietaniphila bkme-9 is able to degrade dehydroabietic acid (dha) via ring hydroxylation by a novel dioxygenase. the dita1, dita2, and dita3 genes, which encode the alpha and beta subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. the ferredoxin gene is 9. 2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditc. a tn5 insertion in the alpha subunit gene, dita1 ...199910217753
adhesion of acinetobacter venetianus to diesel fuel droplets studied with in situ electrochemical and molecular probesthe adhesion of a recently described species, acinetobacter venetianus ve-c3 (f. di cello, m. pepi, f. baldi, and r. fani, res. microbiol. 148:237-249, 1997), to diesel fuel (a mixture of c12 to c28 n-alkanes) and n-hexadecane was studied and compared to that of acinetobacter sp. strain rag-1, which is known to excrete the emulsifying lipopolysaccharide, emulsan. oxygen consumption rates, biomass, cell hydrophobicity, electrophoretic mobility, and zeta potential were measured for the two strains ...199910223998
isolation and characterization of the cis-trans-unsaturated fatty acid isomerase of pseudomonas oleovorans gpo12.pseudomonas oleovorans contains an isomerase which catalyzes the cis-trans conversion of the abundant unsaturated membrane fatty acids 9-cis-hexadecenoic acid (palmitoleic acid) and 11-cis-octadecenoic acid (vaccenic acid). we purified the isomerase from the periplasmic fraction of pseudomonas oleovorans. the molecular mass of the enzyme was estimated to be 80 kda under denaturing conditions and 70 kda under native conditions, suggesting a monomeric structure of the active enzyme. n-terminal seq ...199910322030
an alkane-responsive expression system for the production of fine chemicalsmembrane-located monooxygenase systems, such as the pseudomonas putida mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. in order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the oct plasmid-located alk genes of pseudomonas oleovorans gpo1, a very att ...199910347009
type 2 nadh dehydrogenases in the cyanobacterium synechocystis sp. strain pcc 6803 are involved in regulation rather than respiration.analysis of the genome of synechocystis sp. strain pcc 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 nad(p)h dehydrogenases (ndh-2). the sequence similarity between the translated open reading frames and ndh-2s from other organisms is low, generally not exceeding 30% identity. however, nad(p)h and flavin adenine dinucleotide binding motifs are conserved in all three putative ndh-2s in synechocystis sp. strain pcc 6803. the three open reading fram ...199910383967
physiological adaptations involved in alkane assimilation at a low temperature by rhodococcus sp. strain q15.we examined physiological adaptations which allow the psychrotroph rhodococcus sp. strain q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). during growth at 5 degrees c on hexadecane or diesel fuel, strain q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. a transmission electron microscopy examination of strai ...199910388690
isolation and characterization of a sulfate-reducing bacterium that anaerobically degrades alkanes.an alkane-degrading, sulfate-reducing bacterial strain, ak-01, was isolated from an estuarine sediment with a history of chronic petroleum contamination. the bacterium is a short, nonmotile, non-spore-forming, gram-negative rod. it is mesophilic and grows optimally at ph 6.9 to 7.0 and at an nacl concentration of 1%. formate, fatty acids (c4 to c16) and hydrogen were readily utilized as electron donors. sulfate, sulfite, and thiosulfate were used as electron acceptors, but sulfur, nitrite, and n ...199910388691
fabg, an nadph-dependent 3-ketoacyl reductase of pseudomonas aeruginosa, provides precursors for medium-chain-length poly-3-hydroxyalkanoate biosynthesis in escherichia coli.escherichia coli hosts expressing fabg of pseudomonas aeruginosa showed 3-ketoacyl coenzyme a (coa) reductase activity toward r-3-hydroxyoctanoyl-coa. furthermore, e. coli recombinants carrying the poly-3-hydroxyalkanoate (pha) polymerase-encoding gene phac in addition to fabg accumulated medium-chain-length phas (mcl-phas) from alkanoates. when e. coli fadb or fada mutants, which are deficient in steps downstream or upstream of the 3-ketoacyl-coa formation step during beta-oxidation, respective ...200010781572
identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in amycolatopsis mediterranei dsm5908.seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, amycolatopsis mediterranei dsm5908, by a reverse-cloning approach and characterized. using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a dna fragment of 9,882 bp. of the identified open reading frames, three (oxya to -c) showed significant sequence similarities to ...199910390204
epoxidation of 1,7-octadiene by pseudomonas oleovorans in a membrane bioreactora growing cell culture of pseudomonas oleovorans was used to biotransform 1,7-octadiene to 1,2-epoxy-7,8-octene in a continuous-flow bioreactor with an external membrane module. a dense silicone rubber membrane was used to contact an organic phase, containing both the reactant (1,7-octadiene) and the growth substrate (heptane), with an aqueous biomedium phase containing the biocatalyst. heptane and octadiene delivery to the aqueous phase, and epoxide extraction into the solvent, occurred by diff ...199910397816
integrated two-liquid phase bioconversion and product-recovery processes for the oxidation of alkanes: process design and economic evaluationpseudomonas oleovorans and recombinant strains containing the alkane oxidation genes can produce alkane oxidation products in two-liquid phase bioreactor systems. in these bioprocesses the cells, which grow in the aqueous phase, oxidize apolar, non-water soluble substrates. the apolar products typically accumulate in the emulsified apolar phase. we have studied both the bioconversion systems and several downstream processing systems to separate and purify alkanols from these two-liquid phase med ...199910397885
engineering of stable recombinant bacteria for production of chiral medium-chain-length poly-3-hydroxyalkanoates.in order to scale up medium-chain-length polyhydroxyalkanoate (mcl-pha) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phac1, which encodes one of the mcl-pha polymerases of pseudomonas oleovorans. we used the mini-tn5 system as a tool to construct escherichia coli 193mc1 and p. oleovorans pomc1, which had stable antibiotic resistance and pha production phenotypes when they were cultured i ...199910427005
biosynthesis of polyhydroxyalkanoates from low-rank coal liquefaction products by pseudomonas oleovorans and rhodococcus rubera screening identified several bacteria that were able to use chemically heterogeneous low-rank coal liquefaction products as complex carbon sources for growth. pseudomonas oleovorans and rhodococcus ruber accumulated polyhydroxyalkanoic acids (pha) amounting to 2%-8% of the cell dry weight when the cells were cultivated on these liquefaction products in the absence of any other carbon source. r. ruber accumulated, in addition to pha, small amounts of triacylglycerols. the accumulated pha consis ...199910461375
poly-3-hydroxybutyrate degradation in rhizobium (sinorhizobium) meliloti: isolation and characterization of a gene encoding 3-hydroxybutyrate dehydrogenase.we have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdha) from rhizobium (sinorhizobium) meliloti. the gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pi 6.07). the r. meliloti bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdha is the first gene transcribed in an operon that also includes xdha, encoding xanthine oxidase/dehydrogenase. transc ...19999922248
phaf, a polyhydroxyalkanoate-granule-associated protein of pseudomonas oleovorans gpo1 involved in the regulatory expression system for pha genes.the phac1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl pha) synthase of pseudomonas oleovorans gpo1, which produces mcl pha when grown in an excess of carbon source and under nitrogen limitation. in this work, we have demonstrated, by constructing a recombinant p. oleovorans strain carrying a phac1::lacz reporter system, that the phac1 gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon ...19999922249
screening, nucleotide sequence, and biochemical characterization of an esterase from pseudomonas fluorescens with high activity towards lactones.a genomic library of pseudomonas fluorescens dsm 50106 in a lambdaresiii phage vector was screened in escherichia coli k-12 for esterase activity by using alpha-naphthyl acetate and fast blue rr. a 3.2-kb dna fragment was subcloned from an esterase-positive clone and completely sequenced. esterase estf1 was encoded by a 999-bp open reading frame (orf) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. the deduced amino acid sequences of two other ...19999925571
analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from ralstonia eutropha: generation of beta-alanine auxotrophic tn5 mutants and cloning of the pand gene region.the postulated posttranslational modification of the polyhydroxybutyrate (pha) synthase from ralstonia eutropha by 4-phosphopantetheine was investigated. four beta-alanine auxotrophic tn5-induced mutants of r. eutropha hf39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the pand gene from escherichia coli, encoding l-aspartate-1-decarboxylase (ec 4.1.1.15), whereas two other insertions were mapped in an open reading frame (orf) with strong simila ...199910049372
the palkbfghjkl promoter is under carbon catabolite repression control in pseudomonas oleovorans but not in escherichia coli alk+ recombinants.the alk genes are located on the oct plasmid of pseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. we have investigated the influence of alternative carbon sources on the induction of this pathway in p. oleovorans and escherichia coli alk+ recombinants. in doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. specifically, synthesis of the ...199910049394
polyhydroxyalkanoates degradation affects survival of pseudomonas oleovorans in river water microcosms.bacterial survival in natural environments involves the ability of scavenging nutrients and energy sources. polyhydroxyalkanoates (phas) are intracellular polymers that endow bacteria with enhanced survival capabilities in adverse environmental conditions. in this paper we compared survival of pseudomonas oleovorans wild type and pha depolymerase mutant strains in natural river waters by using microcosms. experiments were performed with water samples collected from the rio de la plata. the survi ...199910615683
the ntrb and ntrc genes are involved in the regulation of poly-3-hydroxybutyrate biosynthesis by ammonia in azospirillum brasilense sp7.azospirillum brasilense sp7 and its ntra (rpon), ntrbc, and ntrc mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (phb) accumulation in media with high and low ammonia concentrations. it was observed that the ntrbc and ntrc mutants can produce phb in both low- and high-c/n-ratio media, while no significant phb production was observed for the wild type or the ntra mutant in low-c/n-ratio media. further investigation by fermentation analysis indicated that the ntrbc and ...200010618211
catabolite repression control by crc in 2xyt medium is mediated by posttranscriptional regulation of bkdr expression in pseudomonas putida.the effect of growth in 2xyt medium on catabolite repression control in pseudomonas putida has been investigated using the bkd operon, encoding branched-chain keto acid dehydrogenase. crc (catabolite repression control protein) was shown to be responsible for repression of bkd operon transcription in 2xyt. bkdr levels were elevated in a p. putida crc mutant, but bkdr transcript levels were the same in both wild type and crc mutant. this suggests that the mechanism of catabolite repression contro ...200010648543
a novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in escherichia coli.a new pathway to synthesize poly(hydroxyalkanoic acids) (pha) was constructed by simultaneously expressing butyrate kinase (buk) and phosphotransbutyrylase (ptb) genes of clostridium acetobutylicum and the two pha synthase genes (phae and phac) of thiocapsa pfennigii in escherichia coli. the four genes were cloned into the bamhi and ecori sites of pbr322, and the resulting hybrid plasmid, pbpp1, conferred activities of all three enzymes to e. coli jm109. cells of this recombinant strain accumula ...200010653745
a positive feedback mechanism controls expression of alks, the transcriptional regulator of the pseudomonas oleovorans alkane degradation pathway.the alks regulator, encoded by the alks gene of the pseudomonas oleovorans oct plasmid, activates the expression of a set of enzymes that allow assimilation of alkanes. we show that the alks protein regulates, both negatively and positively, the expression of its own gene. in the absence of alkanes, alks is expressed from promoter palks1, which is recognized by sigmas-rna polymerase, and whose activity is very low in the exponential phase of growth and considerably higher in stationary phase. al ...200010692156
inactivation of isocitrate lyase leads to increased production of medium-chain-length poly(3-hydroxyalkanoates) in pseudomonas putida.medium-chain-length (mcl) poly(3-hydroxyalkanoates) (phas) are storage polymers that are produced from various substrates and accumulate in pseudomonas strains belonging to rrna homology group i. in experiments aimed at increasing pha production in pseudomonas strains, we generated an mcl pha-overproducing mutant of pseudomonas putida kt2442 by transposon mutagenesis, in which the acea gene was knocked out. this mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isoci ...200010698750
production of polyhydroxyalkanoic acids by ralstonia eutropha and pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production.screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. ralstonia eutropha h16 and pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (pha). r. eutropha and p. oleovorans accumulated pha amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in minera ...200010709978
expression, stability and performance of the three-component alkane mono-oxygenase of pseudomonas oleovorans in escherichia coli.we tested the synthesis and in vivo function of the inducible alkane hydroxylase of pseudomonas oleovorans gpo1 in several escherichia coli recombinants. the enzyme components (alkb, alkg and alkt) were synthesized at various rates in different e. coli hosts, which after induction produced between twofold and tenfold more of the alk components than did p. oleovorans. the enzyme components were less stable in recombinant e. coli hosts than in p. oleovorans. in addition, the specific activity of t ...200010727934
physiological analysis of the expression of the styrene degradation gene cluster in pseudomonas fluorescens st.the effects of different carbon sources on expression of the styrene catabolism genes in pseudomonas fluorescens st were analyzed by using a promoter probe vector, ppr9tt, which contains transcription terminators upstream and downstream of the beta-galactosidase reporter system. expression of the promoter of the stysr operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. this was confirmed by the results of an ...200010742204
properties of engineered poly-3-hydroxyalkanoates produced in recombinant escherichia coli strains.to prepare medium-chain-length poly-3-hydroxyalkanoates (phas) with altered physical properties, we generated recombinant escherichia coli strains that synthesized phas with altered monomer compositions. experiments with different substrates (fatty acids with different chain lengths) or different e. coli hosts failed to produce phas with altered physical properties. therefore, we engineered a new potential pha synthetic pathway, in which ketoacyl-coenzyme a (coa) intermediates derived from the b ...200010742205
poly(3-hydroxyvalerate) depolymerase of pseudomonas lemoignei.pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (pha) depolymerase structural genes (phaz1 to phaz5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (phb), poly(3-hydroxyvalerate) (phv), and related polyesters consisting of short-chain-length hxdroxyalkanoates (pha(scl)) as the sole sources of carbon and energy. four genes (phaz1, phaz2, phaz3, and phaz5) encode phb depolymerases c, b, d, and a, respectively. it was speculated that the remain ...200010742216
xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudocumene to corresponding alcohols, aldehydes, and acids in escherichia coli jm101.xylene monooxygenase of pseudomonas putida mt-2 catalyzes the methylgroup hydroxylation of toluene and xylenes. to investigate the potential of xylene monooxygenase to catalyze multistep oxidations of one methyl group, we tested recombinant escherichia coli expressing the monooxygenase genes xylm and xyla under the control of the alk regulatory system of pseudomonas oleovorans gpo1. expression of xylene monooxygenase genes could efficiently be controlled by n-octane and dicyclopropylketone. xyle ...200010744688
enterotoxigenic escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles.escherichia coli and other gram-negative bacteria produce outer membrane vesicles during normal growth. vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells. enterotoxigenic e. coli (etec) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation. vesicle protein profiles were similar but not identical to outer membranes and differed ...200010777535
toluene monooxygenase-catalyzed epoxidation of alkenes.several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. each of the wild-type organisms degraded all of the alkenes that were tested. epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. a strain of escherichia coli expressing the cloned toluene-4-monooxygenase (t4mo) of pseudomonas mendocina kr1 was able to oxidize butene, butadiene, pentene, and ...200010788354
phag-mediated synthesis of poly(3-hydroxyalkanoates) consisting of medium-chain-length constituents from nonrelated carbon sources in recombinant pseudomonas fragi.recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxy-alkanoate) consisting of medium-chain-length constituents (c(6) to c(14)) (pha(mcl)), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:coa transacylase (phag), has been identified in pseudomonas putida (b. h. a. rehm, n. krüger, and a. steinbüchel, j. biol. chem. 273:24044-24051, 1998). to establish this pha-biosynthetic pathway in a non-pha-accumulating bacterium, we functionally c ...200010788390
gene structures and regulation of the alkane hydroxylase complex in acinetobacter sp. strain m-1.in the long-chain n-alkane degrader acinetobacter sp. strain m-1, two alkane hydroxylase complexes are switched by controlling the expression of two n-alkane hydroxylase-encoding genes in response to the chain length of n-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively.200111160120
cloning and expression of ntnd, encoding a novel nad(p)(+)-independent 4-nitrobenzyl alcohol dehydrogenase from pseudomonas sp. strain tw3.pseudomonas sp. strain tw3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the tol plasmids. we report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnd) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. the gene is located downstream of the previously reported ntn gene cluster. ntnd bear ...200010809692
differential effects of permeating and nonpermeating solutes on the fatty acid composition of pseudomonas putida.we examined the effect of reduced water availability on the fatty acid composition of pseudomonas putida strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes nacl or polyethylene glycol (peg) with a molecular weight of 200 (peg 200) or the nonpermeating solute peg 8000. transmission electron microscopy showed that -1.0-mpa peg 8000-treated cells had convoluted outer membranes, whereas -1.0-mpa nacl-treated or control cells did not. at the ran ...200010831419
effects exerted by transcriptional regulator pcau from acinetobacter sp. strain adp1.protocatechuate degradation is accomplished in a multistep inducible catabolic pathway in acinetobacter sp. strain adp1. the induction is brought about by the transcriptional regulator pcau in concert with the inducer protocatechuate. pcau, a member of the new iclr family of transcriptional regulators, was shown to play a role in the activation of transcription at the promoter for the structural pca genes, leaving open the participation of additional activators. in this work we show that there i ...200111208784
cyanobacterial sulfide-quinone reductase: cloning and heterologous expression.the gene encoding sulfide-quinone reductase (sqr; e.c.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the rhodobacter capsulatus sqr and in an open reading frame found in the genome of aquifex aeolicus. sqr activity was also detected in the unicellular cyanobacterium aphanothece halophytica following sulfide inductio ...200010852862
development of an improved in vitro activity assay for medium chain length pha polymerases based on coenzymea release measurements.an improved activity assay for polyhydroxyalkanoate (pha) polymerases from pseudomonas oleovorans gpo1 was developed. the activity assay is based on the detection of released coenzyme a (coa) using 5,5'-dithiobis (2-nitrobenzoic acid) (dtnb), a compound which specifically reacts with thiol groups. the formed adduct was measured spectrophotometrically with high sensitivity and accuracy. the assay was used to study the effect of several additives on the activity of granule-associated pha polymeras ...200010856771
intracellular depolymerase activity in isolated inclusion bodies containing polyhydroxyalkanoates with long alkyl and functional substituents in the side chain.the in vitro degradation of isolated pseudomonas oleovorans inclusion bodies containing either poly-3-hydroxynonanoate (phn), or poly(-3-hydroxy-5-phenylvalerate) (phpv), or a mixture of these two polymers was investigated. when incubated at 30 degrees c and ph 9, inclusion bodies containing either polyhydroxyoctanoate (pho), phn or phpv exhibited similar degradation rates of approximately 0.94 (+/- 3%) mg/h. the phn and phpv components for inclusion bodies containing a mixture of phn and phpv s ...199910517528
recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin.a novel process for the purification of active medium-chain-length-polyhydroxyalkanoate (mcl-pha) polymerase was developed. this process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation with ion-exchange resin. the mcl-pha polymerase 1 from pseudomonas oleovorans was overproduced from the palk promoter. most of the polymerase produced was sequestered in the cytoplasm as an inactive form in insoluble aggregates. by incubating the protein aggregates w ...200010880359
accumulation of poly[(r)-3-hydroxyalkanoates] in pseudomonas oleovorans during growth with octanoate in continuous culture at different dilution rates.pseudomonas oleovorans atcc 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (c(0)/n(0) ratio). at all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with nh(4)(+) in excess at low c(0)/n(0) ratios, (ii) purely nitrogen-limited growth conditions at high c(0)/n(0) ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-lim ...200010919799
the alkane hydroxylase gene of burkholderia cepacia rr10 is under catabolite repression control.in many microorganisms the first step for alkane degradation is the terminal oxidation of the molecule by an alkane hydroxylase. we report the characterization of a gene coding for an alkane hydroxylase in a burkholderia cepacia strain isolated from an oil-contaminated site. the protein encoded showed similarity to other known or predicted bacterial alkane hydroxylases, although it clustered on a separate branch together with the predicted alkane hydroxylase of a mycobacterium tuberculosis strai ...200111418560
isolation and characterization of diverse halobenzoate-degrading denitrifying bacteria from soils and sediments.denitrifying bacteria capable of degrading halobenzoates were isolated from various geographical and ecological sites. the strains were isolated after initial enrichment on one of the monofluoro-, monochloro-, or monobromo-benzoate isomers with nitrate as an electron acceptor, yielding a total of 33 strains isolated from the different halobenzoate-utilizing enrichment cultures. each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor. the isolates obtai ...200010919805
distribution of alkb genes within n-alkane-degrading bacteria.fifty-four bacterial strains belonging to 37 species were tested for their ability to assimilate short chain and/or medium chain liquid n-alkanes. a gene probe derived from the alkb gene of pseudomonas oleovorans atcc 29347 was utilized in hybridization experiments. results of southern hybridization of pcr-amplificates were compared with those of colony hybridization and dot blot hybridization. strongest signals were received only from gram-negative bacteria growing solely with short n-alkanes ( ...200010971768
role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (pha) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (phag)-mediated pathway for pha biosynthesis in escherichia coli.since pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (pha) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from p. aeruginosa with respect to biosynthesis of phas and rhamnolipids. all isogenic faba, fabb, fabi, rhlg, and phag mutants from p. aeruginosa showed decreased pha accumulation and rhamnolipid production. in the phag (encoding transacylase) mutant rhamnolipid production was o ...200111425728
isolation and characterization of a soluble nadph-dependent fe(iii) reductase from geobacter sulfurreducens.nadph is an intermediate in the oxidation of organic compounds coupled to fe(iii) reduction in geobacter species, but fe(iii) reduction with nadph as the electron donor has not been studied in these organisms. crude extracts of geobacter sulfurreducens catalyzed the nadph-dependent reduction of fe(iii)-nitrilotriacetic acid (nta). the responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. its specific activity for f ...200111443080
mobilization of poly(3-hydroxybutyrate) in ralstonia eutropha.ralstonia eutropha h16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (phb) in the absence of an exogenous carbon source and used the degradation products for growth and survival. isolated native phb granules of mobilized r. eutropha cells released 3-hydroxybutyrate (3hb) at a threefold higher rate than did control granules of nonmobilized bacteria. no 3hb was released by native phb granules of recombinant escherichia coli expressing the phb biosynthetic genes. native phb gr ...200011004196
characterization, seasonal occurrence, and diel fluctuation of poly(hydroxyalkanoate) in photosynthetic microbial mats.in situ poly(hydroxyalkanoate) (pha) levels and repeating-unit compositions were examined in stratified photosynthetic microbial mats from great sippewissett salt marsh, mass., and ebro delta, spain. unlike what has been observed in pure cultures of phototrophic bacteria, the prevalence of hydroxyvalerate (hv) repeating units relative to hydroxybutyrate (hb) repeating units was striking. in the cyanobacteria-dominated green material of sippewissett mats, the mole percent ratio of repeating units ...200011010871
identification and analysis of the polyhydroxyalkanoate-specific beta-ketothiolase and acetoacetyl coenzyme a reductase genes in the cyanobacterium synechocystis sp. strain pcc6803.synechocystis sp. strain pcc6803 possesses a polyhydroxyalkanoate (pha)-specific beta-ketothiolase encoded by phaa(syn) and an acetoacetyl-coenzyme a (coa) reductase encoded by phab(syn). a similarity search of the entire synechocystis genome sequence identified a cluster of two putative open reading frames (orfs) for these genes, slr1993 and slr1994. sequence analysis showed that the orfs encode proteins having 409 and 240 amino acids, respectively. the two orfs are colinear and most probably c ...200011010896
phenylacetyl-coenzyme a is the true inducer of the phenylacetic acid catabolism pathway in pseudomonas putida u.aerobic degradation of phenylacetic acid in pseudomonas putida u is carried out by a central catabolism pathway (phenylacetyl-coenzyme a [coa] catabolon core). induction of this route was analyzed by using different mutants specifically designed for this objective. our results revealed that the true inducer molecule is phenylacetyl-coa and not other structurally or catabolically related aromatic compounds.200011010921
bioconversion of hydrophobic compounds in a continuous closed-gas-loop bioreactor: feasibility assessment and epoxide production.microorganisms can be used as catalysts to produce organic compounds in a highly chemo-, regio- and enantioselective manner, and whole cells do not require the costly addition of cofactors for redox reactions. however, bioconversions are slow compared to alternative chemical reactions, and the biocatalyst works at its best in an aqueous medium, while the transformations of interest frequently involve compounds with a low-aqueous solubility and that are toxic to microorganisms. this results in lo ...200011042552
analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes.degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. two alternative pathways have been described to degrade these fatty acids. one pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme a (coa) reductase and delta(3)-delta(2)-enoyl-coa isomerase, whereas the second involves the epimerization of r-3-hydroxyacyl-coa via a 3-hydroxyacyl-coa epimerase or th ...200011080293
two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates).pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (pha) most effectively at growth rates below the maximum specific growth rate. under adequate conditions, pha accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. for pha production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. it offers production of cells at a specific growth rate in ...200111084589
accumulation of polyhydroxyalkanoic acid containing large amounts of unsaturated monomers in pseudomonas fluorescens bm07 utilizing saccharides and its inhibition by 2-bromooctanoic acid.a psychrotrophic bacterium, pseudomonas fluorescens bm07, which is able to accumulate polyhydroxyalkanoic acid (pha) containing large amounts of 3-hydroxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. when it was grown on heptanoic acid (c(7)) to hexadecanoic acid (c(16)) as the sole carbon source, the monomer compositional characteristics of the synthe ...200111679314
thermostable nadp(+)-dependent medium-chain alcohol dehydrogenase from acinetobacter sp. strain m-1: purification and characterization and gene expression in escherichia coli.nadph-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from acinetobacter sp. strain m-1 was purified and characterized. the purified enzyme had molecular masses of 40 kda as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kda as determined by gel filtration chromatography. the enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from c(2) to c(14) and several ...200011097895
cloning of an intracellular poly[d(-)-3-hydroxybutyrate] depolymerase gene from ralstonia eutropha h16 and characterization of the gene product.an intracellular poly[d(-)-3-hydroxybutyrate] (phb) depolymerase gene (phaz) has been cloned from ralstonia eutropha h16 by the shotgun method, sequenced, and characterized. nucleotide sequence analysis of a 2.3-kbp dna fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 da. the crude extract of escherichia coli containing the phb depolymerase gene digested artificial amorphous phb granules and released mainly oligo ...200111114905
homologous functional expression of cryptic phag from pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway.various pseudomonads are capable of the synthesis of polyhydroxyalkanoate (pha), composed of medium chain length (mcl) 3-hydroxy fatty acids (c6-c14), when grown on simple carbon sources such as, for example, gluconate or acetate. in pseudomonas putida, the fatty acid de novo synthesis and pha synthesis are linked by the transacylase phag. southern hybridization experiments with digoxigenin-labeled phag(pp) from p. putida and genomic dna from various pseudomonads indicate that phag homologues ar ...200011131392
membrane-associated quinoprotein formaldehyde dehydrogenase from methylococcus capsulatus bath.a membrane-associated, dye-linked formaldehyde dehydrogenase (dl-faldh) was isolated from the obligate methylotroph methylococcus capsulatus bath. the enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. soluble nad(p)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane mon ...200111698372
identification of a novel metabolite in the degradation of pyrene by mycobacterium sp. strain ap1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons.mycobacterium sp. strain ap1 grew with pyrene as a sole source of carbon and energy. the identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its c-4, c-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene ...200111722898
polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of pseudomonas oleovorans in natural water microcosms.pseudomonas oleovorans gpo1 and its polyhydroxyalkanoic acid (pha) depolymerization-minus mutant, gpo500 phaz, residing in natural water microcosms, were utilized to asses the effect of pha availability on survival and resistance to stress agents. the wild-type strain showed increased survival compared to the pha depolymerase-minus strain. the appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mu ...200111133449
rubredoxins involved in alkane oxidation.rubredoxins (rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems. several rd genes associated with alkane hydroxylase or rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on n-alkanes (alk-rds). complementation tests in an escherichia coli recombinant containing all pseudomonas putida gpo1 genes necessary for growth on alkanes except rd 2 (alkg) and sequence comparisons showed that the alk-rds can be divi ...200211872724
accumulation of poly[(r)-3-hydroxyalkanoates] in pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources.pseudomonas oleovorans (atcc 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. the growth medium for batch cultures was adjusted such that nitrogen (nh(4)(+)) limitation terminated the exponential-growth phase. during batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(r)-3-hydroxyalkanoates] (mcl-ph ...200111135197
five-gene cluster in clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type a flavoprotein- high-molecular-weight rubredoxin.a five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium clostridium thermoaceticum (moorella thermoacetica) was cloned and sequenced. based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fpra (type a flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). northern blot analysis demonstrated that the five-gene clust ...200111160086
two-iron rubredoxin of pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, cd and nmr spectroscopies.a minigene encoding the c-terminal domain of the 2fe rubredoxin of pseudomonas oleovorans was created from the parental alk g gene contained in the expression plasmid pkk223-3. the vector directed the high-level production of the c-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1fe form. the 1fe form of the c-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolu ...200111171083
molecular screening for alkane hydroxylase genes in gram-negative and gram-positive strains.we have developed highly degenerate oligonucleotides for polymerase chain reaction (pcr) amplification of genes related to the pseudomonas oleovorans gpo1 and acinetobacter sp. adp1 alkane hydroxylases, based on a number of highly conserved sequence motifs. in all gram-negative and in two out of three gram-positive strains able to grow on medium- (c6-c11) or long-chain n-alkanes (c12-c16), pcr products of the expected size were obtained. the pcr fragments were cloned and sequenced and found to e ...199911207749
the black cat/white cat principle of signal integration in bacterial promoters. 200111226149
hyporesponsiveness of spret/ei mice to lethal shock induced by tumor necrosis factor and implications for a tnf-based antitumor therapy.tumor necrosis factor (tnf) is a central mediator in lethal shock and an interesting cytokine for anticancer therapy. to inhibit tnf-induced lethal shock, it is important to identify protective genes. here we demonstrate that the spret/ei mouse strain, derived from mus spretus, exhibits an extremely dominant resistance to tnf-induced lethal inflammation. an interspecific backcross experiment revealed that the tnf hyporesponse is linked to loci on chromosomes 2, 6, and 11. treatment of inoculated ...200212089334
the role of the fatty acid beta-oxidation multienzyme complex from pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadba operon from p. oleovorans and of the enoyl-coa hydratase genes phaj from p. oleovorans and pseudomonas putida.in order to investigate the role of the putative epimerase function of the beta-oxidation multienzyme complex (fadba) in the provision of (r)-3-hydroxyacyl-coa thioesters for medium-chain-length polyhydroxyalkanoate (pha(mcl)) biosynthesis, the fadba(po) operon of pseudomonas oleovorans was cloned and characterized. the fadba(po) operon and a class-ii pha synthase gene of pseudomonas aeruginosa were heterologously co-expressed in escherichia coli to determine whether the putative epimerase funct ...200212115060
detection of genes for alkane and naphthalene catabolism in rhodococcus sp. strain 1bn.rhodococcus sp. 1bn was isolated from a contaminated site and showed various biodegradative capabilities. besides naphthalene, strain 1bn degraded medium- (c6) and long-chain alkanes (c16-c28), benzene and toluene, alone or when the hydrocarbons were mixed in equal proportions. the nucleotide sequence of an alk polymerase chain reaction (pcr) fragment revealed a 59% nucleotide homology to the pseudomonas oleovorans alkb gene. the nar fragments were highly homologous to genes coding for large and ...200011233165
poly-beta-hydroxybutyrate biosynthesis in the facultative methylotroph methylobacterium extorquens am1: identification and mutation of gap11, gap20, and phar.methylobacterium extorquens am1, a serine cycle facultative methylotroph, accumulates poly-beta-hydroxybutyrate (phb) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates. recently, the identification and mutation of the genes involved in the biosynthesis and degradation of phb have been described for this bacterium, demonstrating that two of the genes of the phb cycle (phaa and phab) are also involved in c(1) and c(2) metabolism, as part of a n ...200212399487
mechanism of cis-trans isomerization of unsaturated fatty acids in pseudomonas putida.we studied the pattern of the cis-trans isomerization of unsaturated fatty acids in cells of pseudomonas putida s12 grown in a medium supplemented with oleic acid which was deuterated at both of the c atoms of its double bond. direct evidence that isomerization does not include a transient saturation of the double bond was obtained. in addition, analysis of the amino acid sequences of the seven known cti proteins identified them as heme-containing proteins of the cytochrome c type.200312591893
succession of phenotypic, genotypic, and metabolic community characteristics during in vitro bioslurry treatment of polycyclic aromatic hydrocarbon-contaminated sediments.dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (pahs) was removed from the milwaukee confined disposal facility and examined for in situ biodegradative capacity. molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. ester-linked phospholipid fatty acids (plfa), multiplex pcr of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, gen ...200111282603
response of electrically stimulated cells of pseudomonas oleovorans strain atcc 29347 suspended in silicone oil.a high intensity direct current was applied for more than 10 min onto a bacterial suspension of pseudomonas oleovorans atcc 29347 suspended in silicone oil. the application of a gradually increased electric field from 0 to 2500 v x cm(-1) resulted in a decrease of the optical density of the bacterial suspension and the occurrence of a peak current of several hundred microa for living cells instead of a linear increase (few microa) for killed or lyophilised cells. this procedure is not only a rap ...200111356578
analysis of pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.the pseudomonas putida gpo1 (commonly known as pseudomonas oleovorans gpo1) alkbfghjkl and alkst gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the oct plasmid, separated by 9.7 kb of dna. this dna segment encodes, amongst others, a methyl-accepting transducer protein (alkn) that may be involved in chemotaxis to alkanes. in p. putida p1, the alkbfghjkl and alkst gene clusters are flanked by almost identical copies of the ins ...200111390693
alteration of chain length substrate specificity of aeromonas caviae r-enantiomer-specific enoyl-coenzyme a hydratase through site-directed mutagenesis.aeromonas caviae r-specific enoyl-coenzyme a (enoyl-coa) hydratase (phaj(ac)) is capable of providing (r)-3-hydroxyacyl-coa with a chain length of four to six carbon atoms from the fatty acid beta-oxidation pathway for polyhydroxyalkanoate (pha) synthesis. in this study, amino acid substitutions were introduced into phaj(ac) by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. a crystallographic structure analysis of ...200312902277
long-chain aldehyde dehydrogenase that participates in n-alkane utilization and wax ester synthesis in acinetobacter sp. strain m-1.a long-chain aldehyde dehydrogenase, ald1, was found in a soluble fraction of acinetobacter sp. strain m-1 cells grown on n-hexadecane as a sole carbon source. the gene (ald1) was cloned from the chromosomal dna of the bacterium. the open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. the ald1 gene was stably expresse ...200010919810
identification and characterization of a new enoyl coenzyme a hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant escherichia coli.the biosynthetic pathway of medium-chain-length (mcl) polyhydroxyalkanoates (phas) from fatty acids has been established in fadb mutant escherichia coli strain by expressing the mcl-pha synthase gene. however, the enzymes that are responsible for the generation of (r)-3-hydroxyacyl coenzyme a (r3ha-coas), the substrates for pha synthase, have not been thoroughly elucidated. escherichia coli maoc, which is homologous to pseudomonas aeruginosa (r)-specific enoyl-coa hydratase (phaj1), was identifi ...200312949091
oxidation of soluble oil emulsions and emulsifiers by pseudomonas oleovorans and pseudomonas formicans. 195613355396
prediction of reduction potential changes in rubredoxin: a molecular mechanics approach.predicting the effects of mutation on the reduction potential of proteins is crucial in understanding how reduction potentials are modulated by the protein environment. previously, we proposed that an alanine vs. a valine at residue 44 leads to a 50-mv difference in reduction potential found in homologous rubredoxins because of a shift in the polar backbone relative to the iron site due to the different side-chain sizes. here, the aim is to determine the effects of mutations to glycine, isoleuci ...200314581187
role of phad in accumulation of medium-chain-length poly(3-hydroxyalkanoates) in pseudomonas oleovorans.pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (phas) as intracellular storage material. to analyze the possible involvement of phad in medium-chain-length (mcl) pha biosynthesis, we generated a phad knockout mutant by homologous recombination. upon disruption of the phad gene, mcl pha polymer accumulation was decreased. the pha granule size was reduced, and the number of granules inside the cell was increased. furthermore, mutant cells appeared to be smaller than wild- ...200010966380
carbon limitation induces sigma(s)-dependent gene expression in pseudomonas fluorescens in soil.recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to pseudomonas are not severely limited in bulk soil. indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. we show that a plasmid (pgm115)-borne transcriptional fusion between the sigma(s)-dependent escherichia coli promoter p(fic) and lacz functions as a reliable reporter for carbon availability in pseudomonas f ...200111472905
coexpression of genetically engineered 3-ketoacyl-acp synthase iii (fabh) and polyhydroxyalkanoate synthase (phac) genes leads to short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production from glucose in escherichia coli jm109.polyhydroxyalkanoates (phas) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. short-chain-length (scl) phas consist of monomer units of c3 to c5, medium-chain-length (mcl) phas consist of monomer units of c6 to c14, and scl-mcl phas consist of monomers ranging in size from c4 to c14. although previous studies using recombinant escherichia coli have shown that either scl or mcl pha polymers could be produced from glucose, this study presents t ...200414766582
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