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S Harayama

Publications and source records attributed to S Harayama.

At least 73 records · Page 4Linked to original sources
PubMed1997

Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7.

2-Carboxybenzaldehyde dehydrogenase from the phenanthrene-degrading bacterium Nocardioides sp. strain KP7 was purified and characterized. The purified enzyme had a molecular mass of 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 205 kDa by gel filtration chromatography. Thus, the homotetramer of the 53-kDa subunit constituted an active enzyme. The apparent Km and kcat values of this enzyme for 2-carboxybenzaldehyde were 100 microM and 39 s(-1), respectively, and those for NAD+ were 83 microM and 32 s(-1), respectively. The structural gene for this enzyme was cloned and sequenced. The length of the gene was 1,455 bp. The nucleotide sequence of the 10,279 bp of DNA around the gene for 2-carboxybenzaldehyde dehydrogenase was also determined, and seven open reading frames were found in this DNA region. These were the genes for 1-hydroxy-2-naphthoate dioxygenase (phdI) and trans-2'-carboxybenzalpyruvate aldolase (phdJ), orf1, the gene for 2-carboxybenzaldehyde dehydrogenase (phdK), orf2/orf3, and orf4. The amino acid sequence of the orf1 product was similar to that of the aromatic hydrocarbon transporter gene (pcaK) in Pseudomonas putida PRS2000. The amino acid sequence of the orf4 product revealed a similarity to cytochrome P-450 proteins. The region between phdK and orf4 encoded orf2 and orf3 on different strands. The amino acid sequences of the orf2 and orf3 products exhibited no significant similarity to the reported sequences in protein databases.

Actinomycetales↗
PubMed1996

Cloning, sequencing and sites of expression of genes for the hydroxyarginine-containing adhesive-plaque protein of the mussel Mytilus galloprovincialis.

A segment of Mytilus galloprovincialis foot protein 3 (Mgfp-3) cDNA was amplified by means of reverse-transcription (RT)/PCR with degenerate primers. The 5' and 3' regions of the cloned segment were amplified by means of rapid amplification of cDNA ends. The 5'-region clones had almost identical nucleotide sequences, but two sequences were found among 3'-region clones. The Mgfp-3 coding region was amplified between a 5' untranslated sequence and one of two 3' untranslated sequences. Two cDNA clones which encoded variants Mgfp-3A and Mgfp-3B, were isolated. These two clones encoded proteins with 70 and 77 amino acid residues, of which the first 24 residues are predicted to be signal peptides. The existence of additional variants was suggested by the sequences of other clones. Thus, it was suggested that Mgfp-3 genes constitute a gene family. RT/PCR of RNA from developing larvae indicates that Mgfp-3 genes are transcribed after settlement. RT/PCR of RNA from major organs and in situ hybridization of the foot indicate that Mgfp-3 genes are transcribed in a limited part of the foot, i.e., the phenol gland and a distal part of the accessory gland.

Amino Acid Sequence↗
PubMed1996

Adhesive protein cDNA sequence of the mussel Mytilus coruscus and its evolutionary implications.

cDNA encoding the adhesive protein of the mussel Mytilus coruscus (Mcfp1) was isolated. The coding region encoded 848 amino acids (a.a.) comprising the 20-a.a. signal peptide, the 21-a.a. nonrepetitive linker, and the 805-a.a. repetitive domain. Although the first 204 nucleotides and the 3'-untranslated region of Mcfp1 cDNA were homologous to corresponding parts of M. galloprovincialis adhesive protein (Mgfp1) cDNA, the other parts diverged. The representative repeat motif of the repetitive domain, YKPK(I/P)(S/T)YPP(T/S), was similar but slightly different from the repeat motif of Mgfp1. The codon usage patterns for the same amino acids were different in different positions of the decapeptide motif. Almost identical nucleotide sequences encoding the two to 13 repeats appeared several times in the repetitive region, which suggests that the adhesive protein genes of mussels have evolved through the duplication of these repeat units.

Amino Acid Sequence↗
PubMed1996

Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products.

Partial nucleotide sequences of the gyrB genes (DNA gyrase B subunit genes) of 15 Acinetobacter strains, including the type and reference strains of genomic species 1 to 12 (A. calcoaceticus [genomic species 1], A. baumannii [genomic species 2], Acinetobacter genomic species 3, A. haemolyticus [genomic species 4], A. junii [genomic species 5], Acinetobacter genomic species 6, A. johnsonii [genomic species 7], A. lwoffii [genomic species 8], Acinetobacter genomic species 9, Acinetobacter genomic species 10, Acinetobacter genomic species 11, and A. radioresistens [genomic species 12]), were determined by sequencing the PCR-amplified fragments of gyrB. The gyrB sequence homology among these Acinetobacter strains ranged from 69.6 to 99.7%. A phylogenetic analysis, using the gyrB sequences, indicates that genomic species 1, 2, and 3 formed one cluster (87.3 to 90.3% identity), while genomic species 8 and 9 formed another cluster (99.7% identity). These results are consistent with those of DNA-DNA hybridization and of biochemical systematics. On the other hand, the topology of the published phylogenetic tree based on the 16S rRNA sequences of the Acinetobacter strains was quite different from that of the gyrB-based tree. The numbers of substitution in the 16S rRNA gene sequences were not high enough to construct a reliable phylogenetic tree. The gyrB-based analysis indicates that the genus Acinetobacter is highly diverse and that a reclassification of this genus would be required.

Acinetobacter↗
PubMed1996

Characteristics of transposon insertion mutants of mandelic acid-utilizing Pseudomonas putida strain A10L.

A soil isolate, Pseudomonas putida strain A10L that utilizes mandelate via the mandelate pathway was mutagenized by transposon Tn5-Mob insertion and mutant 168 lacking mandelate racemase (MR) and a mutant 254 lacking benzoylformate decarboxylase (BFDC) were obtained. Expression of (S)-mandelate dehydrogenase (MDH), BFDC, NAD(+)-dependent benzaldehyde dehydrogenase (BDH) and NADP(+)-dependent BDH in the MR-lacking mutant was not affected by the insertion, and it was inducible similarly to the wild type strain. On the other hand, expression of MR and MDH in the BFDC-lacking mutant was low and constitutive, and NAD(+)- and NADP(+)-dependent BDHs were produced at a rather high level under non-induced conditions by the mutant. Genes for MR (mdlA), MDH (mdlB), and BFDC (mdlC) were indicated to be organized in an operon in the order of mdlCBA. Optical resolution to obtain (R)-mandelate, a useful synthon for pharmaceuticals, was shown to be performed with the MR-lacking mutant.

Aldehyde Oxidoreductases↗
PubMed1996

Cloning and characterization of the gene encoding pyrroloquinoline quinone-dependent poly(vinyl alcohol) dehydrogenase of Pseudomonas sp. strain VM15C.

A gene library of poly(vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca2+, and Mg2+ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.

Alcohol Oxidoreductases↗
PubMed1995

Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida.

The substrate specificities of two catechol 2,3-dioxygenases, one encoded by xylE on the TOL plasmid pWW0 and the other encoded by nahH on the NAH7 plasmid, were investigated. The XylE catechol 2,3-dioxygenase catalyzes the ring-cleavage of catechol, 3-methylcatechol and 4-methylcatechol. The NahH catechol 2,3-dioxygenase was partially deficient in oxidizing 3-methylcatechol due to defects in two catalytic properties. First, NahH has a lower kcat value for 3-methylcatechol compared to XylE, and secondly, NahH is more susceptible than XylE to suicide inhibition by 3-methylcatechol. To identify the amino acid residues of XylE and NahH responsible for the differences in the efficacy of the 3-methylcatechol oxidation, kcat and kinact (the rate constant for suicide inhibition) for 3-methylcatechol were determined for several NahH-XylE hybrid proteins, each of which consisted of the NahH sequence in the N-terminal region and the XylE sequence in the C-terminal region. It is shown that a single amino acid substitution present in the NahH sequence, His250-->Gln, was responsible for the reduced kcat and increased kinact values for 3-methylcatechol. In addition to the substitution at residue 250, some substitution(s) at residues 77-102 were responsible for the twofold difference in the kinact values for NahH and XylE with 3-methylcatechol. We also show that the binding site of 3-methylcatechol for suicide inhibition is different from the catalytic site.

Amino Acid Sequence↗
PubMed1995

Biochemical characterization of a Ca(2+)-dependent lectin from the hemolymph of a photosymbiotic marine bivalve, Tridacna derasa (Röding).

A Ca(2+)-dependent lectin was purified from the hemolymph of a photosymbiotic bivalve, Tridacna derasa. An electrophoretically homogeneous form was obtained by using affinity chromatography with Sepharose 4B. More than 80% of the hemolymph protein was accounted for by this lectin. The apparent molecular mass of the lectin, in its native form exhibiting hemagglutinating activity, was estimated by gel filtration analysis to be approximately 480 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in the absence of reductants, it migrated as a single band corresponding to a very large size, while in the presence of 2-mercaptoethanol, it migrated as two distinct bands of 23 and 46 kDa. These results indicate that the subunits were linked by disulfide bridges to form the native protein. After reducing pyridylethylation, each of the 23- and 46-kDa polypeptides was isolated by gel filtration in a mobile phase containing guanidine-HCl. The two polypeptides had the same amino-terminal sequence and a similar amino-acid composition, and in the presence of 2-mercaptoethanol gave a single band on isoelectric focusing at a pH of 6.0. The results suggested that the 46-kDa peptide is a homodimer of 23-kDa subunits held together by a covalent bond other than a disulfide linkage. This lectin required calcium ions for its activity. By ultraviolet spectrophotometry the association constant for the calcium ion was determined to be 0.88 mM. The hemagglutinating activity decreased dramatically below pH 6.5, but re-increased to the original level when the solution was neutralized. Such a pH-dependent alteration of the ligand-binding activity was similar to that found in vertebrate asialoglycoprotein receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Purification and characterization of Acinetobacter calcoaceticus 4-hydroxybenzoate 3-hydroxylase after its overexpression in Escherichia coli.

4-Hydroxybenzoate 3-hydroxylase [EC 1.14.13.2] from Acinetobacter calcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pobA) in Escherichia coli. Overexpression was accomplished by placing the folA gene (encoding trimethoprim-resistant dihydrofolate reductase) directly downstream of the pobA gene, and demanding growth of recombinants on elevated concentration of trimethoprim. Presumably, the surviving variants have undergone a genetic alteration which allowed the overexpression of both folA and pobA. 4-Hydroxybenzoate 3-hydroxylase was purified in two chromatographic steps, characterized biochemically, and its properties were compared to those of its homolog from Pseudomonas fluorescens. The two enzymes differ in their response to Cl- ion inhibition. A single amino acid change in the putative NADPH-binding site is proposed to account for this difference. The inhibitory and catalytic properties of substrate analogs were also examined.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1995

PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains.

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5' end which allow direct sequencing of amplified PCR products. With these primers, DNA segments of the predicted size were amplified from a variety of gram-negative and gram-positive genera. The nucleotide sequences of the amplified gyrB DNA from three P. putida strains were determined directly from the amplified fragments. The base substitution frequency of gyrB between the strains of P. putida was much higher than that of the 16S rRNA gene. With a specific set of PCR primers, it was possible to amplify gyrB fragments selectively from P. putida or its subgroups. The direct sequencing method of gyrB developed in this study provides a rapid and convenient system for bacterial identification, taxonomic analysis, and monitoring of bacteria in the natural environment.

Amino Acid Sequence↗
PubMed1995

Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida.

Two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylC and xylG genes, respectively, on TOL plasmid pWW0 of Pseudomonas putida. The nucleotide sequence of xylC was determined in this study. A protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of P. putida (pWW0) (J. P. Shaw and S. Harayama, Eur. J. Biochem. 191:705-714, 1990); however, the amino-terminal sequence of this protein does not correspond to that predicted from the xylC sequence but does correspond to that predicted from the xylG sequence. The protein purified in the earlier work was therefore 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product). This conclusion was confirmed by the fact that this protein oxidized 2-hydroxymuconic semialdehyde (kcat/Km = 1.6 x 10(6) s-1 M-1) more efficiently than benzaldehyde (kcat/Km = 3.2 x 10(4) s-1 M-1). The xylC product, the genuine benzaldehyde dehydrogenase, was purified from extracts of P. putida (pWW0-161 delta rylG) which does not synthesize 2-hydroxymuconic semialdehyde dehydrogenase. The amino-terminal sequence of the purified protein corresponds to the amino-terminal sequence deduced from the xylC sequence. This enzyme efficiently oxidized benzaldehyde (kcat/Km = 1.7 x 10(7) s-1 M-1) and its analogs but did not oxidize 2-hydroxymuconic semialdehyde or its analogs.

Alcohol Oxidoreductases↗
PubMed1995

Sequential enrichment of microbial populations exhibiting enhanced biodegradation of crude oil.

The distribution of oil-degrading bacteria in the coastal water and sediments of Hokkaido, Japan, was surveyed. The potential of mixed microbial populations to degrade weathered crude oil was not confined to any ecological components (water or sediment) nor to the sampling stations. One microbial culture that was stable during repeated subculturing degraded 45% of the saturates and 20% of the aromatics present in crude oil in 10 days during the initial screening. The residual hydrocarbons in this culture were extracted by chloroform and dispersed in a fresh seawater-based medium and subsequently inoculated with microorganisms from the first culture. After full growth of the second culture, the residual hydrocarbons were again extracted and dispersed in a fresh medium in which microorganisms from the second culture had been inoculated. This sequential process was carried out six times to enrich those microorganisms that grew on the recalcitrant components of crude oil. After repeated exposure of the residual crude oil to the enriched microorganisms, about 80% of the initially added crude oil was degraded. The cultures obtained after each enrichment cycle were kept, and the degradation of fresh crude oil by the enriched microorganisms was examined. The degradative activity of the enriched cultures increased as the number of enrichment cycles increased. A microbial population that had been selected six times on the residual crude oil could degrade 70% of the saturates and 30% of the aromatics of crude oil. Thus, growth of a microbial population on residual crude oil improved its ability to biodegrade crude oil.

Bacteria↗
PubMed1995

Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus.

Variation in the adhesive protein gene sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus collected in Delaware, Kamaishi (Japan), and Alaska, respectively, was analyzed by the polymerase chain reaction (PCR) using two sets of oligonucleotide primers. The first set, Me 13 and Me 14, was designed to amplify the repetitive region. The length of the amplified fragments was highly variable, even among samples of the same species. Another set, Me 15 and Me 16, was designed to amplify a part of the nonrepetitive region. The length of the amplified fragments was uniform in each species and differed interspecifically; 180, 168, and 126 bp for M. edulis, M. trossulus, and M. galloprovincialis, respectively. The amplified sequence of M. trossulus resembled that of M. edulis. Mussels from other sites were also examined by PCR using Me 15 and Me 16. Wild mussels from Tromsö (Norway) and cultured mussels from Brittany (France) were identified as M. edulis. Cultured mussels from the Mediterranean coast of France and wild mussels from Shimizu (Japan) were identified as M. galloprovincialis. Some wild mussels from Hiura (Japan) were identified as a hybrid between M. galloprovincialis and M. trossulus. Thus, the length of this part (variable region) of the sequence is proposed as a diagnostic marker for these three morphologically similar species and their hybrids.

Amino Acid Sequence↗
PubMed1994

Regulation of the rpoN, ORF102 and ORF154 genes in Pseudomonas putida.

The DNA sequence downstream of the Pseudomonas putida rpoN gene and the adjacent ORF102 was determined. This region encodes an ORF (ORF154) whose gene product was found to be homologous to a family of phosphotransferases. Insertional mutagenesis and analysis of mRNA transcripts showed that the rpoN gene is transcribed separately from the two downstream genes. The rpoN promoter was localized to an 86 nucleotide-long region upstream of the rpoN gene by examination of the expression of a series of rpoN::lacZ fusions. The expression of rpoN in P. putida was independent of the nitrogen status of the cell but was 5 times higher in the rpoN mutant than in the wild-type strain, suggesting that the expression of the rpoN gene in this organism is autoregulated.

Amino Acid Sequence↗
PubMed1994

Codon usage patterns suggest independent evolution of two catabolic operons on toluene-degradative plasmid TOL pWW0 of Pseudomonas putida.

TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes responsible for the degradation of toluene. The structural genes for these catobolic enzymes are clustered into two operons--namely, the xy/CMAB and xy/XYZLTEGFJQKIH operons. We examined the codon usage patterns of these catabolic genes by measuring the codon-usage distances between pairs of these catabolic genes. The codon-usage distance, d, between gene 1 and gene 2 was defined as d = [sigma(pj-qj)2]1/2, are the frequencies of the j-th codon in gene 1 and 2, respectively, j being any one of the 64 possible codons. We found that the genes in the same operon exhibit similar codon-usage patterns while genes in the different operons exhibit different codon bias. This observation suggests that genes in the same operon have coevolved, and that the ancestors of the xy/CMAB and xy/XYZLTEGFJQKIH operons evolved in different organisms.

Biodegradation, Environmental↗
PubMed1994

Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth.

Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.

Ascorbic Acid↗
PubMed1994

Conversion of pBR322-based plasmids into broad-host-range vectors by using the Tn3 transposition mechanism.

We constructed a series of transposon vectors which allow efficient in vitro gene manipulation and subsequent introduction of cloned DNA into a variety of gram-negative bacteria. Transfer of the cloned fragment from these multicopy plasmids into self-transmissible broad-host-range vectors is achieved in vivo, using the Tn3 transposition mechanism. Transposition into a variety of broad-host-range plasmids proceeds efficiently, and the resulting recombinant plasmids can be readily transferred and maintained in a variety of gram-negative bacteria. The utility of the transposable vectors was demonstrated by the introduction and expression of the lacIPOZY sequences of Escherichia coli into Pseudomonas putida strains, allowing them to utilize lactose as a sole source of carbon and energy.

Alkanes↗