Tsuda M

References (43)

Title : Intestinal Permeability of Drugs in Caco-2 Cells Cultured in Microfluidic Devices - Sasaki_2022_Biol.Pharm.Bull_45_1246
Author(s) : Sasaki Y , Tatsuoka H , Tsuda M , Sumi T , Eguchi Y , So K , Higuchi Y , Takayama K , Torisawa Y , Yamashita F
Ref : Biol Pharm Bull , 45 :1246 , 2022
Abstract : Microfluidic devices are attracting attention for their ability to provide a biomimetic microenvironment wherein cells are arranged in a particular pattern and provided fluidic and mechanical forces. In this study, we evaluated drug transport across Caco-2 cell layers in microfluidic devices and investigated the effects of fluid flow on drug transport and metabolism. We designed a microfluidic device that comprises two blocks of polydimethylsiloxane and a sandwiched polyethylene terephthalate membrane with pores 3.0 microm in diameter. When cultured in a dynamic fluid environment, Caco-2 cells were multilayered and developed microvilli on the surface as compared with a static environment. Drugs with higher lipophilicity exhibited higher permeability across the Caco-2 layers, as well as in the conventional method using Transwells, and the fluidic conditions had little effect on permeability. In the Caco-2 cell layers cultured in Transwells and microfluidic devices, the basal-to-apical transport of rhodamine 123, a substrate of P-glycoprotein, was greater than the apical-to-basal transport, and the presence of tariquidar, an inhibitor of P-glycoprotein, completely diminished asymmetric transport. Furthermore, fluidic conditions promoted the metabolism of temocapril by carboxylesterases. On the other hand, we showed that fluidic conditions have little effect on gene expression of several transporters and metabolic enzymes. These results provide useful information regarding the application of microfluidic devices in drug transport and metabolism studies.
ESTHER : Sasaki_2022_Biol.Pharm.Bull_45_1246
PubMedSearch : Sasaki_2022_Biol.Pharm.Bull_45_1246
PubMedID: 36047192

Title : Non-neuronal cardiac cholinergic system influences CNS via the vagus nerve to acquire a stress-refractory propensity - Oikawa_2016_Clin.Sci.(Lond)_130_1913
Author(s) : Oikawa S , Kai Y , Tsuda M , Ohata H , Mano A , Mizoguchi N , Sugama S , Nemoto T , Suzuki K , Kurabayashi A , Muramoto K , Kaneda M , Kakinuma Y
Ref : Clinical Science (Lond) , 130 :1913 , 2016
Abstract : We previously developed cardiac ventricle-specific choline acetyltransferase (ChAT) gene-overexpressing transgenic mice (ChAT tgm), i.e. an in vivo model of the cardiac non-neuronal acetylcholine (NNA) system or non-neuronal cardiac cholinergic system (NNCCS). By using this murine model, we determined that this system was responsible for characteristics of resistance to ischaemia, or hypoxia, via the modulation of cellular energy metabolism and angiogenesis. In line with our previous study, neuronal ChAT-immunoreactivity in the ChAT tgm brains was not altered from that in the wild-type (WT) mice brains; in contrast, the ChAT tgm hearts were the organs with the highest expression of the ChAT transgene. ChAT tgm showed specific traits in a central nervous system (CNS) phenotype, including decreased response to restraint stress, less depressive-like and anxiety-like behaviours and anti-convulsive effects, all of which may benefit the heart. These phenotypes, induced by the activation of cardiac NNCCS, were dependent on the vagus nerve, because vagus nerve stimulation (VS) in WT mice also evoked phenotypes similar to those of ChAT tgm, which display higher vagus nerve discharge frequency; in contrast, lateral vagotomy attenuated these traits in ChAT tgm to levels observed in WT mice. Furthermore, ChAT tgm induced several biomarkers of VS responsible for anti-convulsive and anti-depressive-like effects. These results suggest that the augmentation of the NNCCS transduces an effective and beneficial signal to the afferent pathway, which mimics VS. Therefore, the present study supports our hypothesis that activation of the NNCCS modifies CNS to a more stress-resistant state through vagus nerve activity.
ESTHER : Oikawa_2016_Clin.Sci.(Lond)_130_1913
PubMedSearch : Oikawa_2016_Clin.Sci.(Lond)_130_1913
PubMedID: 27528769

Title : Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome - Anda_2015_Proc.Natl.Acad.Sci.U.S.A_112_14343
Author(s) : Anda M , Ohtsubo Y , Okubo T , Sugawara M , Nagata Y , Tsuda M , Minamisawa K , Mitsui H
Ref : Proc Natl Acad Sci U S A , 112 :14343 , 2015
Abstract : rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.
ESTHER : Anda_2015_Proc.Natl.Acad.Sci.U.S.A_112_14343
PubMedSearch : Anda_2015_Proc.Natl.Acad.Sci.U.S.A_112_14343
PubMedID: 26534993
Gene_locus related to this paper: 9rhiz-a0a0s2en26 , 9rhiz-a0a0p0z830 , 9rhiz-a0a0p0z878 , 9rhiz-a0a0p0z9k0 , 9rhiz-a0a0s2epz8

Title : Complete Genome Sequence of Polypropylene Glycol- and Polyethylene Glycol-Degrading Sphingopyxis macrogoltabida Strain EY-1 - Ohtsubo_2015_Genome.Announc_3_e01399
Author(s) : Ohtsubo Y , Nagata Y , Numata M , Tsuchikane K , Hosoyama A , Yamazoe A , Tsuda M , Fujita N , Kawai F
Ref : Genome Announc , 3 : , 2015
Abstract : Strain EY-1 was isolated from a microbial consortium growing on a random polymer of ethylene oxide and propylene oxide. Strain EY-1 grew on polyethylene glycol and polypropylene glycol and identified as Sphingopyxis macrogoltabida. Here, we report the complete genome sequence of Sphingopyxis macrogoltabida EY-1. The genome of strain EY-1 is comprised of a 4.76-Mb circular chromosome, and five plasmids. The whole finishing was conducted in silico, with aids of computational tools GenoFinisher and AceFileViewer. Strain EY-1 is available from Biological Resource Center, National Institute of Technology and Evaluation (Tokyo, Japan) (NITE).
ESTHER : Ohtsubo_2015_Genome.Announc_3_e01399
PubMedSearch : Ohtsubo_2015_Genome.Announc_3_e01399
PubMedID: 26634754
Gene_locus related to this paper: sphmc-a0a0n9ujk2

Title : Properties and biotechnological applications of natural and engineered haloalkane dehalogenases - Nagata_2015_Appl.Microbiol.Biotechnol_99_9865
Author(s) : Nagata Y , Ohtsubo Y , Tsuda M
Ref : Applied Microbiology & Biotechnology , 99 :9865 , 2015
Abstract : Haloalkane dehalogenases (HLDs) convert halogenated compounds to corresponding alcohols, halides, and protons. They belong to alpha/beta-hydrolases, and their principal catalytic mechanism is SN2 nucleophilic substitution followed by the addition of water. Since HLDs generally have broad and different substrate specificities, they have various biotechnological applications. HLDs have previously been believed to be present only in bacterial strains that utilize xenobiotic halogenated compounds, and three archetypal HLDs, i.e., DhlA, DhaA, and LinB, have been intensively investigated by biochemical, structural, and computational analyses. Furthermore, by using the resulting data and target-selected random mutagenesis approaches, these HLDs have been successfully engineered to improve their substrate specificities and activities. In addition, important insights into protein evolution have been obtained by studying these HLDs. At the same time, the genome and metagenome information has revealed that HLD homologues are widely distributed in many bacterial strains, including ones that have not been reported to degrade halogenated compounds. Some of these cryptic HLD homologues have been experimentally confirmed to be "true" HLDs with unique substrate specificities and enantioselectivities. Although their biological functions and physiological roles remain mysterious, these potential HLDs are considered promising materials for the development of new biocatalysts.
ESTHER : Nagata_2015_Appl.Microbiol.Biotechnol_99_9865
PubMedSearch : Nagata_2015_Appl.Microbiol.Biotechnol_99_9865
PubMedID: 26373728

Title : Complete Genome Sequence of a Phenanthrene Degrader, Mycobacterium sp. Strain EPa45 (NBRC 110737), Isolated from a Phenanthrene-Degrading Consortium - Kato_2015_Genome.Announc_3_e00782
Author(s) : Kato H , Ogawa N , Ohtsubo Y , Oshima K , Toyoda A , Mori H , Nagata Y , Kurokawa K , Hattori M , Fujiyama A , Tsuda M
Ref : Genome Announc , 3 : , 2015
Abstract : A phenanthrene degrader, Mycobacterium sp. EPa45, was isolated from a phenanthrene-degrading consortium. Here, we report the complete genome sequence of EPa45, which has a 6.2-Mb single circular chromosome. We propose a phenanthrene degradation pathway in EPa45 based on the complete genome sequence.
ESTHER : Kato_2015_Genome.Announc_3_e00782
PubMedSearch : Kato_2015_Genome.Announc_3_e00782
PubMedID: 26184940
Gene_locus related to this paper: 9myco-a0a0g3iim6 , 9myco-a0a0g3ijm3 , 9myco-a0a0g3ipv9 , 9myco-a0a0g3iuf8 , 9myco-a0a0g3ivy5 , 9myco-a0a0g3ihb1

Title : Complete Genome Sequence of Polyvinyl Alcohol-Degrading Strain Sphingopyxis sp. 113P3 (NBRC 111507) - Ohtsubo_2015_Genome.Announc_3_e01169
Author(s) : Ohtsubo Y , Nagata Y , Numata M , Tsuchikane K , Hosoyama A , Yamazoe A , Tsuda M , Fujita N , Kawai F
Ref : Genome Announc , 3 :e01169 , 2015
Abstract : Strain 113P3 was isolated from activated sludge and identified as a polyvinyl alcohol (PVA)-degrading Pseudomonas species; it was later reidentified as Sphingopyxis species. Only three genes are directly relevant to the metabolism of PVA and comprise the pva operon, which was deposited as accession no. AB190228. Here, we report the complete genome sequence of strain 113P3, which has been conserved as a stock culture (NBRC 111507) at the Biological Resource Center, National Institute of Technology and Evaluation (NITE) (Tokyo, Japan). The genome of strain 113P3 is composed of a 4.4-Mb circular chromosome and a 243-kb plasmid. The whole finishing was conducted in silico except for four PCRs. The sequence corresponding to AB190288 exists on the chromosome.
ESTHER : Ohtsubo_2015_Genome.Announc_3_e01169
PubMedSearch : Ohtsubo_2015_Genome.Announc_3_e01169
PubMedID: 26472829
Gene_locus related to this paper: sphs1-a0a0m4cze0

Title : Complete Genome Sequence of the Thermophilic Polychlorinated Biphenyl Degrader Geobacillus sp. Strain JF8 (NBRC 109937) - Shintani_2014_Genome.Announc_2_e01213
Author(s) : Shintani M , Ohtsubo Y , Fukuda K , Hosoyama A , Ohji S , Yamazoe A , Fujita N , Nagata Y , Tsuda M , Hatta T , Kimbara K
Ref : Genome Announc , 2 : , 2014
Abstract : Geobacillus sp. strain JF8 (NBRC 109937) utilizes biphenyl and naphthalene as sole carbon sources and degrades polychlorinated biphenyl (PCB) at 60 degrees C. Here, we report the complete nucleotide sequence of the JF8 genome (a 3,446,630-bp chromosome and a 39,678-bp plasmid). JF8 has the smallest genome among the known PCB degraders.
ESTHER : Shintani_2014_Genome.Announc_2_e01213
PubMedSearch : Shintani_2014_Genome.Announc_2_e01213
PubMedID: 24459274
Gene_locus related to this paper: geotn-a4isp0 , 9baci-s5yvc0 , 9baci-s5yzj8

Title : Complete Genome Sequence of Pseudomonas aeruginosa MTB-1, Isolated from a Microbial Community Enriched by the Technical Formulation of Hexachlorocyclohexane - Ohtsubo_2014_Genome.Announc_2_e01130
Author(s) : Ohtsubo Y , Sato T , Kishida K , Tabata M , Ogura Y , Hayashi T , Tsuda M , Nagata Y
Ref : Genome Announc , 2 : , 2014
Abstract : Pseudomonas aeruginosa MTB-1 does not degrade gamma-hexachlorocyclohexane (gamma-HCH), but this bacterium persistently coexists with a gamma-HCH-degrading strain, Sphingomonas sp. MM-1, in a microbial community enriched by the technical formulation of HCH. Here we report the complete MTB-1 genome sequence, with a 6.6-Mb circular chromosome.
ESTHER : Ohtsubo_2014_Genome.Announc_2_e01130
PubMedSearch : Ohtsubo_2014_Genome.Announc_2_e01130
PubMedID: 24459257
Gene_locus related to this paper: pseae-PA3695 , pseae-PA5080 , pseae-q9i252

Title : Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards beta-hexachlorocyclohexane - Moriuchi_2014_AMB.Express_4_72
Author(s) : Moriuchi R , Tanaka H , Nikawadori Y , Ishitsuka M , Ito M , Ohtsubo Y , Tsuda M , Damborsky J , Prokop Z , Nagata Y
Ref : AMB Express , 4 :72 , 2014
Abstract : Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI's catalytic performance towards beta-hexachlorocyclohexane (beta-HCH) is considerably higher than LinBUT's. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT's beta-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.
ESTHER : Moriuchi_2014_AMB.Express_4_72
PubMedSearch : Moriuchi_2014_AMB.Express_4_72
PubMedID: 25401073

Title : Complete Genome Sequence of Pseudomonas sp. Strain TKP, Isolated from a gamma-Hexachlorocyclohexane-Degrading Mixed Culture - Ohtsubo_2014_Genome.Announc_2_e01241
Author(s) : Ohtsubo Y , Kishida K , Sato T , Tabata M , Kawasumi T , Ogura Y , Hayashi T , Tsuda M , Nagata Y
Ref : Genome Announc , 2 : , 2014
Abstract : Pseudomonas sp. strain TKP does not degrade gamma-hexachlorocyclohexane (gamma-HCH), but it persistently coexists with the gamma-HCH-degrading Sphingobium sp. strain TKS in a mixed culture enriched by gamma-HCH. Here, we report the complete genome sequence of strain TKP, which consists of one circular chromosome with a size of 7 Mb.
ESTHER : Ohtsubo_2014_Genome.Announc_2_e01241
PubMedSearch : Ohtsubo_2014_Genome.Announc_2_e01241
PubMedID: 24482516
Gene_locus related to this paper: psefl-e2xn15 , psefs-c3k632 , psefs-laaa , psefs-c3k813 , psefl-e2xkc8 , 9psed-v9qxq1 , 9psed-v9qr47

Title : Complete Genome Sequence of the gamma-Hexachlorocyclohexane-Degrading Bacterium Sphingomonas sp. Strain MM-1 - Tabata_2013_Genome.Announc_1_e00247
Author(s) : Tabata M , Ohtsubo Y , Ohhata S , Tsuda M , Nagata Y
Ref : Genome Announc , 1 : , 2013
Abstract : gamma-Hexachlorocyclohexane (gamma-HCH) is a man-made chlorinated insecticide that has caused serious environmental problems. Here, we report the complete genome sequence of the gamma-HCH-degrading bacterium Sphingomonas sp. strain MM-1, which consists of one chromosome and five plasmids. All the specific lin genes that are almost identical to those of Sphingobium japonicum UT26 for the conversion of gamma-HCH to beta-ketoadipate are dispersed on four out of the five plasmids.
ESTHER : Tabata_2013_Genome.Announc_1_e00247
PubMedSearch : Tabata_2013_Genome.Announc_1_e00247
PubMedID: 23682148
Gene_locus related to this paper: 9sphn-m4s0a2 , 9sphn-m4sjq0 , 9sphn-m4s4n0 , 9sphn-m4rwk9 , 9sphn-m4s1q7

Title : Senescent case of cholesterol ester storage disease that progressed to liver cirrhosis with a novel mutation (N250H) of lysosomal acid lipase gene - Kojima_2013_Hepatol.Res_43_1361
Author(s) : Kojima S , Watanabe N , Takashimizu S , Kagawa T , Shiraishi K , Koizumi J , Hirabayashi K , Ohkubo T , Kamiguchi H , Tsuda M , Mine T
Ref : Hepatol Res , 43 :1361 , 2013
Abstract : The patient, a 69-year-old man, had a chief complaint of hepatomegaly. The liver was palpated four fingerbreadths below the costal margin, and the spleen was three fingerbreadths below the costal margin. There were no other abnormal findings. Laparoscopy showed that the liver resembled an orange-yellow crayon in appearance and was nodular. The pathological findings of the liver biopsy tissue were consistent with liver cirrhosis. Inside the fibrous septum was an apparent aggregation of enlarged macrophages that phagocytosed lipid components, as well as enlarged Kupffer cells that phagocytosed lipid droplets. Electron microscopy showed the lipid droplets to have a moth-eaten appearance. Using monocytes extracted from the peripheral blood, acid lipase activity was measured by fluorescence spectrometry using 4-methylumbelliferone palmitate as a substrate. This patient's human lysosomal acid lipase activity was 0.020 nM/min per 10(6) cells, corresponding to 5.9% of that in healthy subjects (0.332 +/- 0.066 nM/min per 10(6) cells). Cholesterol ester storage disease was therefore diagnosed. The acid lipase A base sequence obtained from leukocytes by direct sequencing was compared with a library. This patient had a point mutation of N250H/N250H in exon 7, a novel gene abnormality that has not previously been reported.
ESTHER : Kojima_2013_Hepatol.Res_43_1361
PubMedSearch : Kojima_2013_Hepatol.Res_43_1361
PubMedID: 23675960
Gene_locus related to this paper: human-LIPA

Title : Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205 - Okai_2013_J.Bacteriol_195_2642
Author(s) : Okai M , Ohtsuka J , Imai LF , Mase T , Moriuchi R , Tsuda M , Nagata K , Nagata Y , Tanokura M
Ref : Journal of Bacteriology , 195 :2642 , 2013
Abstract : The enzymes LinBUT and LinBMI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of beta-hexachlorocyclohexane (beta-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinBMI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinBMI were categorized into three groups based on the efficiency of the first-step (from beta-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinBMI and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinBMI. The dynamics simulation analyses of wild-type LinBMI and LinBUT revealed that the entrance of the substrate access tunnel of LinBUT was more flexible than that of LinBMI, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.
ESTHER : Okai_2013_J.Bacteriol_195_2642
PubMedSearch : Okai_2013_J.Bacteriol_195_2642
PubMedID: 23564170
Gene_locus related to this paper: sphpi-q6vqx3

Title : Heart-specific overexpression of choline acetyltransferase gene protects murine heart against ischemia through hypoxia-inducible factor-1alpha-related defense mechanisms - Kakinuma_2013_J.Am.Heart.Assoc_2_e004887
Author(s) : Kakinuma Y , Tsuda M , Okazaki K , Akiyama T , Arikawa M , Noguchi T , Sato T
Ref : J Am Heart Assoc , 2 :e004887 , 2013
Abstract : BACKGROUND: Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium. METHODS AND
RESULTS: To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)-expressing cells and heart-specific ChAT transgenic (ChAT-tg) mice. Compared with cardiomyocytes of wild-type (WT) mice, those of the ChAT-tg mice had high levels of ACh and hypoxia-inducible factor (HIF)-1alpha protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT-overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT-tg mice showed similar hemodynamics; after MI, however, the ChAT-tg mice had better survival than did the WT mice. In the ChAT-tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post-MI remodeling. The ChAT-tg heart was more resistant to ischemia-reperfusion injury than was the WT heart.
CONCLUSIONS: These results suggest that the activated cardiac ACh-HIF-1alpha cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self-defense against ischemia.
ESTHER : Kakinuma_2013_J.Am.Heart.Assoc_2_e004887
PubMedSearch : Kakinuma_2013_J.Am.Heart.Assoc_2_e004887
PubMedID: 23525439

Title : A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.) - Tanaka_2012_BMC.Genomics_13_352
Author(s) : Tanaka Y , Tsuda M , Yasumoto K , Yamagishi H , Terachi T
Ref : BMC Genomics , 13 :352 , 2012
Abstract : BACKGROUND: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. RESULTS: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences. CONCLUSIONS: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.
ESTHER : Tanaka_2012_BMC.Genomics_13_352
PubMedSearch : Tanaka_2012_BMC.Genomics_13_352
PubMedID: 22846596
Gene_locus related to this paper: rapsa-a0a6j0jyp3 , rapsa-a0a6j0lzs2 , rapsa-a0a6j0lba8 , rapsa-a0a6j0lmu0

Title : Complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl degrader - Ohtsubo_2012_J.Bacteriol_194_6970
Author(s) : Ohtsubo Y , Maruyama F , Mitsui H , Nagata Y , Tsuda M
Ref : Journal of Bacteriology , 194 :6970 , 2012
Abstract : We report the complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl-degrading strain isolated from a soil sample in Tokyo. The genome contains a single circular 5,196,935-bp chromosome and no plasmids.
ESTHER : Ohtsubo_2012_J.Bacteriol_194_6970
PubMedSearch : Ohtsubo_2012_J.Bacteriol_194_6970
PubMedID: 23209225
Gene_locus related to this paper: 9burk-k0hvd1 , 9burk-k0hw51 , 9burk-k0hxu4 , 9burk-k0i5a9 , 9burk-k0i8t8 , 9burk-k0i3l8 , 9burk-k0i392

Title : The lin genes for gamma-hexachlorocyclohexane degradation in Sphingomonas sp. MM-1 proved to be dispersed across multiple plasmids - Tabata_2011_Biosci.Biotechnol.Biochem_75_466
Author(s) : Tabata M , Endo R , Ito M , Ohtsubo Y , Kumar A , Tsuda M , Nagata Y
Ref : Biosci Biotechnol Biochem , 75 :466 , 2011
Abstract : A gamma-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of gamma-HCH led to the detection of five gamma-HCH metabolites, gamma-pentachlorocyclohexene, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol, 2,5-dichlorohydroquinone, 1,2,4-trichlorobenzene, and 2,5-dichlorophenol, strongly suggesting that MM-1 has the lin genes for gamma-HCH degradation originally identified in the well-studied gamma-HCH-degrading strain Sphingobium japonicum UT26. Southern blot, PCR amplification, and sequencing analyses indicated that MM-1 has seven lin genes for the conversion of gamma-HCH to beta-ketoadipate (six structural genes, linA to linF, and one regulatory gene, linR). MM-1 carried four plasmids, of 200, 50, 40, and 30 kb. Southern blot analysis revealed that all seven lin genes were dispersed across three of the four plasmids, and that IS6100, often found close to the lin genes, was present on all four plasmids.
ESTHER : Tabata_2011_Biosci.Biotechnol.Biochem_75_466
PubMedSearch : Tabata_2011_Biosci.Biotechnol.Biochem_75_466
PubMedID: 21389627
Gene_locus related to this paper: sphpi-linb

Title : Complete nucleotide sequence of TOL plasmid pDK1 provides evidence for evolutionary history of IncP-7 catabolic plasmids - Yano_2010_J.Bacteriol_192_4337
Author(s) : Yano H , Miyakoshi M , Ohshima K , Tabata M , Nagata Y , Hattori M , Tsuda M
Ref : Journal of Bacteriology , 192 :4337 , 2010
Abstract : To understand the mechanisms for structural diversification of Pseudomonas-derived toluene-catabolic (TOL) plasmids, the complete sequence of a self-transmissible plasmid pDK1 with a size of 128,921 bp from Pseudomonas putida HS1 was determined. Comparative analysis revealed that (i) pDK1 consisted of a 75.6-kb IncP-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pDK1 were highly similar to those of MOB(H) group of mobilizable plasmids, and (iii) the toluene-catabolic (xyl) gene clusters of pDK1 were derived through homologous recombination, transposition, and site-specific recombination from the xyl gene clusters homologous to another TOL plasmid, pWW53. The minireplicons of pDK1 and its related IncP-7 plasmids, pWW53 and pCAR1, that contain replication and partition genes were maintained in all of six Pseudomonas strains tested, but not in alpha- or betaproteobacterial strains. The recipient host range of conjugative transfer of pDK1 was, however, limited to two Pseudomonas strains. These results indicate that IncP-7 plasmids are essentially narrow-host-range and self-transmissible plasmids that encode MOB(H) group-related transfer functions and that the host range of IncP-7-specified conjugative transfer was, unlike the situation in other well-known plasmids, narrower than that of its replication.
ESTHER : Yano_2010_J.Bacteriol_192_4337
PubMedSearch : Yano_2010_J.Bacteriol_192_4337
PubMedID: 20581207

Title : Complete genome sequence of the representative gamma-hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26 - Nagata_2010_J.Bacteriol_192_5852
Author(s) : Nagata Y , Ohtsubo Y , Endo R , Ichikawa N , Ankai A , Oguchi A , Fukui S , Fujita N , Tsuda M
Ref : Journal of Bacteriology , 192 :5852 , 2010
Abstract : Sphingobium japonicum strain UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in gamma-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.
ESTHER : Nagata_2010_J.Bacteriol_192_5852
PubMedSearch : Nagata_2010_J.Bacteriol_192_5852
PubMedID: 20817768
Gene_locus related to this paper: psepa-q6vpf3 , sphju-d4z1p3 , sphju-d4z3u6 , sphpi-linb , sphju-d4yyy8

Title : Novel organization of aromatic degradation pathway genes in a microbial community as revealed by metagenomic analysis - Suenaga_2009_ISME.J_3_1335
Author(s) : Suenaga H , Koyama Y , Miyakoshi M , Miyazaki R , Yano H , Sota M , Ohtsubo Y , Tsuda M , Miyazaki K
Ref : Isme J , 3 :1335 , 2009
Abstract : Several types of environmental bacteria that can aerobically degrade various aromatic compounds have been identified. The catabolic genes in these bacteria have generally been found to form operons, which promote efficient and complete degradation. However, little is known about the degradation pathways in bacteria that are difficult to culture in the laboratory. By functionally screening a metagenomic library created from activated sludge, we had earlier identified 91 fosmid clones carrying genes for extradiol dioxygenase (EDO), a key enzyme in the degradation of aromatic compounds. In this study, we analyzed 38 of these fosmids for the presence and organization of novel genes for aromatics degradation. Only two of the metagenomic clones contained complete degradation pathways similar to those found in known aromatic compound-utilizing bacteria. The rest of the clones contained only subsets of the pathway genes, with novel gene arrangements. A circular 36.7-kb DNA form was assembled from the sequences of clones carrying genes belonging to a novel EDO subfamily. This plasmid-like DNA form, designated pSKYE1, possessed genes for DNA replication and stable maintenance as well as a small set of genes for phenol degradation; the encoded enzymes, phenol hydroxylase and EDO, are capable of the detoxification of aromatic compounds. This gene set was found in 20 of the 38 analyzed clones, suggesting that this 'detoxification apparatus' may be widespread in the environment.
ESTHER : Suenaga_2009_ISME.J_3_1335
PubMedSearch : Suenaga_2009_ISME.J_3_1335
PubMedID: 19587775
Gene_locus related to this paper: 9bact-c6kti0 , 9bact-c6ktz1 , 9bact-c6ku06 , 9bact-c6ku85 , 9bact-c6kub4 , 9bact-c6kud7 , 9bact-c6kwi7 , 9bact-c6l0y5 , 9burk-q0gqt7 , psepu-TDNF

Title : Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate - Pavlova_2009_Nat.Chem.Biol_5_727
Author(s) : Pavlova M , Klvana M , Prokop Z , Chaloupkova R , Banas P , Otyepka M , Wade RC , Tsuda M , Nagata Y , Damborsky J
Ref : Nat Chemical Biology , 5 :727 , 2009
Abstract : Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.
ESTHER : Pavlova_2009_Nat.Chem.Biol_5_727
PubMedSearch : Pavlova_2009_Nat.Chem.Biol_5_727
PubMedID: 19701186

Title : Glucose regulation of dipeptidyl peptidase IV gene expression is mediated by hepatocyte nuclear factor-1alpha in epithelial intestinal cells - Gu_2008_Clin.Exp.Pharmacol.Physiol_35_1433
Author(s) : Gu N , Tsuda M , Matsunaga T , Adachi T , Yasuda K , Ishihara A , Tsuda K
Ref : Clinical & Experimental Pharmacology & Physiology , 35 :1433 , 2008
Abstract : 1. Dipeptidyl peptidase IV (DPP-IV) is a new drug target in the treatment of Type 2 diabetes. Dipeptidyl peptidase IV enzyme activity is significantly altered in Type 2 diabetic patients with hyperglycaemia, but the underlying molecular mechanisms remain unclear. 2. The first aim of the present study was to clarify whether glucose regulates DPP-IV enzyme activity. To address this, DPP-IV gene expression and enzyme activity were measured in Caco2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) concentrations of glucose. We observed that high glucose inhibited DPP-IV gene expression and enzyme activity. 3. The second aim of the present study was to investigate whether hepatocyte nuclear factor (HNF)-1alpha contributes to glucose regulation of DPP-IV gene expression. To explore this question, associations between the gene expression of DPP-IV and HNF-1alpha were examined in Caco-2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) glucose. We found that the pattern of glucose-regulated DPP-IV gene expression is similar to that of HNF-1alpha. Moreover, to elucidate whether glucose regulation of DPP-IV gene expression is affected when HNF-1alpha is inhibited, we produced two stable cell lines in which a dominant-negative mutant HNF-1alphaR271G or basic vectors were stably expressed. We found that glucose regulation of DPP-IV gene expression was compromised in HNF-1alphaR271G cells, but was well maintained in basic vector cells. 4. These results suggest that glucose regulation of DPP-IV gene expression is mediated by HNF-1alpha.
ESTHER : Gu_2008_Clin.Exp.Pharmacol.Physiol_35_1433
PubMedSearch : Gu_2008_Clin.Exp.Pharmacol.Physiol_35_1433
PubMedID: 18671716

Title : Insertion sequence-based cassette PCR: cultivation-independent isolation of gamma-hexachlorocyclohexane-degrading genes from soil DNA - Fuchu_2008_Appl.Microbiol.Biotechnol_79_627
Author(s) : Fuchu G , Ohtsubo Y , Ito M , Miyazaki R , Ono A , Nagata Y , Tsuda M
Ref : Applied Microbiology & Biotechnology , 79 :627 , 2008
Abstract : gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.
ESTHER : Fuchu_2008_Appl.Microbiol.Biotechnol_79_627
PubMedSearch : Fuchu_2008_Appl.Microbiol.Biotechnol_79_627
PubMedID: 18425509
Gene_locus related to this paper: psepa-q6vpf3 , sphpi-linb

Title : The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution - Pavlova_2007_J.Struct.Biol_157_384
Author(s) : Pavlova M , Klvana M , Jesenska A , Prokop Z , Konecna H , Sato T , Tsuda M , Nagata Y , Damborsky J
Ref : J Struct Biol , 157 :384 , 2007
Abstract : Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.
ESTHER : Pavlova_2007_J.Struct.Biol_157_384
PubMedSearch : Pavlova_2007_J.Struct.Biol_157_384
PubMedID: 17084094

Title : Degradation of beta-hexachlorocyclohexane by haloalkane dehalogenase LinB from gamma-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205 - Ito_2007_Arch.Microbiol_188_313
Author(s) : Ito M , Prokop Z , Klvana M , Otsubo Y , Tsuda M , Damborsky J , Nagata Y
Ref : Arch Microbiol , 188 :313 , 2007
Abstract : The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal gamma-isomer and noninsecticidal alpha-, beta-, and delta-isomers, among which beta-HCH is the most recalcitrant and has caused serious environmental problems. A gamma-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded beta-HCH more rapidly than the well-characterized gamma-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, beta-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A haloalkane dehalogenase LinB (LinB(MI)) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB(UT)) was identified as an enzyme responsible for the two-step conversion of beta-HCH to TCDL. This property of LinB(MI) contrasted with that of LinB(UT), which catalyzed only the first step conversion of beta-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB(MI). However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB(MI). Sequence analysis revealed that MI1205 possesses the IS6100-flanked cluster that contains two copies of the linB (MI) gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.
ESTHER : Ito_2007_Arch.Microbiol_188_313
PubMedSearch : Ito_2007_Arch.Microbiol_188_313
PubMedID: 17516046
Gene_locus related to this paper: sphpi-linb

Title : Complete sequence determination combined with analysis of transposition\/site-specific recombination events to explain genetic organization of IncP-7 TOL plasmid pWW53 and related mobile genetic elements - Yano_2007_J.Mol.Biol_369_11
Author(s) : Yano H , Garruto CE , Sota M , Ohtsubo Y , Nagata Y , Zylstra GJ , Williams PA , Tsuda M
Ref : Journal of Molecular Biology , 369 :11 , 2007
Abstract : Recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (TOL) plasmids and their residing transposons. To elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pWW53, a TOL plasmid from Pseudomonas putida MT53. pWW53 was found to belong to the IncP-7 incompatibility group that play important roles in the catabolism of several xenobiotics. pWW53 carried two distinct transposase-resolvase gene clusters (tnpAR modules), five short terminal inverted repeats (IRs), and three site-specific resolution (res) sites that are all typical of class II transposons. This organization of pWW53 suggested the four possible transposable regions, Tn4657 to Tn4660. The largest 86 kb region (Tn4657) spanned the three other regions, and Tn4657 and Tn4660 (62 kb) covered all of the 36 xyl genes for toluene catabolism. Our subsequent transposition experiments clarified that the three transposons, Tn4657 to Tn4659, indeed exhibit their transposability, and that pWW53 also generated another 37 kb toluene-catabolic transposon, Tn4656, which carried the two separated and inversely oriented segments of pWW53: the tnpRA-IR module of Tn4658 and a part of xyl gene clusters on Tn4657. The Tn4658 transposase was able to mediate the transposition of Tn4658, Tn4657, and Tn4656, while the Tn4659 transposase catalyzed only the transposition of Tn4659. Tn4656 was formed by the Tn4658 resolvase-mediated site-specific inversion between the two inversely oriented res sites on pWW53. These findings and comparison with other catabolic plasmids clearly indicate multiple copies of transposition-related genes and sites on one plasmid and their recombination activities contribute greatly to the diversification of plasmid structures as well as wide dissemination of the evolutionary common gene clusters in various plasmids.
ESTHER : Yano_2007_J.Mol.Biol_369_11
PubMedSearch : Yano_2007_J.Mol.Biol_369_11
PubMedID: 17408691

Title : Crystallization and preliminary crystallographic analysis of a haloalkane dehalogenase, DbjA, from Bradyrhizobium japonicum USDA110 - Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
Author(s) : Sato Y , Natsume R , Tsuda M , Damborsky J , Nagata Y , Senda T
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 63 :294 , 2007
Abstract : Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. The haloalkane dehalogenase DbjA constitutes a novel substrate-specificity class with high catalytic activity for beta-methylated haloalkanes. In order to reveal the mechanism of its substrate specificity, DbjA has been crystallized using the hanging-drop vapour-diffusion method. The best crystals were obtained using the microseeding technique with a reservoir solution consisting of 17-19.5%(w/v) PEG 4000, 0.2 M calcium acetate and 0.1 M Tris-HCl pH 7.7-8.0. The space group of the DbjA crystal is P2(1)2(1)2, with unit-cell parameters a = 212.9, b = 117.8, c = 55.8 A. The crystal diffracts to 1.75 A resolution.
ESTHER : Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
PubMedSearch : Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
PubMedID: 17401198

Title : Identification of a response regulator gene for catabolite control from a PCB-degrading beta-proteobacteria, Acidovorax sp. KKS102 - Ohtsubo_2006_Mol.Microbiol_60_1563
Author(s) : Ohtsubo Y , Goto H , Nagata Y , Kudo T , Tsuda M
Ref : Molecular Microbiology , 60 :1563 , 2006
Abstract : Acidovorax sp. (formally Pseudomonas sp.) strain KKS102 carries a bph operon for the degradation of PCB/biphenyl. Transcription from the pE promoter for the bph operon was found to be under catabolite control, i.e. the promoter activity was at a lower level when succinate, fumarate or acetate was added to the culture. Some mutations in the immediate upstream region of the pE promoter resulted in catabolite-insensitive and constitutively low promoter activity, suggesting that a transcriptional activator was involved in catabolite control. A genetic screen for a pE promoter activator identified two tandemly arranged genes, bphP and bphQ, that encoded proteins homologous to the sensor kinases and response regulators, respectively, of two-component regulatory system. In the bphPQ double mutant, pE promoter activity was weak and catabolite-insensitive, and a supply of the bphQ gene alone led to the restoration of the catabolite response. The mechanism of catabolite repression in KKS102 is explained in terms of inhibition of activation by BphQ. The genes highly similar to bphQ were found from several beta-proteobacteria, such as Burkholderia cenocepacia J2315, B. multivorans ATCC17616, B. xenovorans LB400 and Ralstonia solanacearum RS1085.
ESTHER : Ohtsubo_2006_Mol.Microbiol_60_1563
PubMedSearch : Ohtsubo_2006_Mol.Microbiol_60_1563
PubMedID: 16796688

Title : Complete nucleotide sequence of an exogenously isolated plasmid, pLB1, involved in gamma-hexachlorocyclohexane degradation - Miyazaki_2006_Appl.Environ.Microbiol_72_6923
Author(s) : Miyazaki R , Sato Y , Ito M , Ohtsubo Y , Nagata Y , Tsuda M
Ref : Applied Environmental Microbiology , 72 :6923 , 2006
Abstract : The alpha-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, gamma-hexachlorocyclohexane (gamma-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of gamma-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS6100 composite transposon containing two copies of linB, and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other alpha-proteobacterial strains but not to any of the beta- or gamma-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for gamma-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in gamma-HCH degradation.
ESTHER : Miyazaki_2006_Appl.Environ.Microbiol_72_6923
PubMedSearch : Miyazaki_2006_Appl.Environ.Microbiol_72_6923
PubMedID: 16963556
Gene_locus related to this paper: sphpi-linb

Title : Comparative analysis of argK-tox clusters and their flanking regions in phaseolotoxin-producing Pseudomonas syringae pathovars - Genka_2006_J.Mol.Evol_63_401
Author(s) : Genka H , Baba T , Tsuda M , Kanaya S , Mori H , Yoshida T , Noguchi MT , Tsuchiya K , Sawada H
Ref : Journal of Molecular Evolution , 63 :401 , 2006
Abstract : DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181-10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064-11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named "tox islands." The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437-457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands.
ESTHER : Genka_2006_J.Mol.Evol_63_401
PubMedSearch : Genka_2006_J.Mol.Evol_63_401
PubMedID: 16927007
Gene_locus related to this paper: psesy-PSPTO4540

Title : Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase - Sota_2006_J.Bacteriol_188_4057
Author(s) : Sota M , Yano H , Ono A , Miyazaki R , Ishii H , Genka H , Top EM , Tsuda M
Ref : Journal of Bacteriology , 188 :4057 , 2006
Abstract : The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.
ESTHER : Sota_2006_J.Bacteriol_188_4057
PubMedSearch : Sota_2006_J.Bacteriol_188_4057
PubMedID: 16707697
Gene_locus related to this paper: psepu-q1xgk7

Title : Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities - Sato_2005_Appl.Environ.Microbiol_71_4372
Author(s) : Sato Y , Monincova M , Chaloupkova R , Prokop Z , Ohtsubo Y , Minamisawa K , Tsuda M , Damborsky J , Nagata Y
Ref : Applied Environmental Microbiology , 71 :4372 , 2005
Abstract : Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.
ESTHER : Sato_2005_Appl.Environ.Microbiol_71_4372
PubMedSearch : Sato_2005_Appl.Environ.Microbiol_71_4372
PubMedID: 16085827

Title : Recipient range of IncP-7 conjugative plasmid pCAR2 from Pseudomonas putida HS01 is broader than from other Pseudomonas strains - Shintani_2005_Biotechnol.Lett_27_1847
Author(s) : Shintani M , Habe H , Tsuda M , Omori T , Yamane H , Nojiri H
Ref : Biotechnol Lett , 27 :1847 , 2005
Abstract : The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.
ESTHER : Shintani_2005_Biotechnol.Lett_27_1847
PubMedSearch : Shintani_2005_Biotechnol.Lett_27_1847
PubMedID: 16328978
Gene_locus related to this paper: psest-bpdF

Title : Degradation of beta-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 - Nagata_2005_Appl.Environ.Microbiol_71_2183
Author(s) : Nagata Y , Prokop Z , Sato Y , Jerabek P , Kumar A , Ohtsubo Y , Tsuda M , Damborsky J
Ref : Applied Environmental Microbiology , 71 :2183 , 2005
Abstract : Beta-Hexachlorocyclohexane (beta-HCH) is the most recalcitrant among the alpha-, beta-, gamma-, and delta-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of gamma-HCH in Sphingomonas paucimobilis UT26, metabolizes beta-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.
ESTHER : Nagata_2005_Appl.Environ.Microbiol_71_2183
PubMedSearch : Nagata_2005_Appl.Environ.Microbiol_71_2183
PubMedID: 15812056

Title : Identification and characterization of genes involved in the downstream degradation pathway of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 - Endo_2005_J.Bacteriol_187_847
Author(s) : Endo R , Kamakura M , Miyauchi K , Fukuda M , Ohtsubo Y , Tsuda M , Nagata Y
Ref : Journal of Bacteriology , 187 :847 , 2005
Abstract : Sphingomonas paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of gamma-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of gamma-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for gamma-HCH utilization.
ESTHER : Endo_2005_J.Bacteriol_187_847
PubMedSearch : Endo_2005_J.Bacteriol_187_847
PubMedID: 15659662
Gene_locus related to this paper: psepa-q6vqy9

Title : Modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 by engineering of its entrance tunnel - Chaloupkova_2003_J.Biol.Chem_278_52622
Author(s) : Chaloupkova R , Sykorova J , Prokop Z , Jesenska A , Monincova M , Pavlova M , Tsuda M , Nagata Y , Damborsky J
Ref : Journal of Biological Chemistry , 278 :52622 , 2003
Abstract : Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
ESTHER : Chaloupkova_2003_J.Biol.Chem_278_52622
PubMedSearch : Chaloupkova_2003_J.Biol.Chem_278_52622
PubMedID: 14525993

Title : Structure of haloacetate-catabolic IncP-1beta plasmid pUO1 and genetic mobility of its residing haloacetate-catabolic transposon - Sota_2003_J.Bacteriol_185_6741
Author(s) : Sota M , Kawasaki H , Tsuda M
Ref : Journal of Bacteriology , 185 :6741 , 2003
Abstract : The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1beta plasmid R751. Comparison of pUO1 with three other IncP-1beta plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.
ESTHER : Sota_2003_J.Bacteriol_185_6741
PubMedSearch : Sota_2003_J.Bacteriol_185_6741
PubMedID: 14594853
Gene_locus related to this paper: morsp-deh1

Title : Reconstruction of mycobacterial dehalogenase Rv2579 by cumulative mutagenesis of haloalkane dehalogenase LinB - Nagata_2003_Appl.Environ.Microbiol_69_2349
Author(s) : Nagata Y , Prokop Z , Marvanova S , Sykorova J , Monincova M , Tsuda M , Damborsky J
Ref : Applied Environmental Microbiology , 69 :2349 , 2003
Abstract : The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.
ESTHER : Nagata_2003_Appl.Environ.Microbiol_69_2349
PubMedSearch : Nagata_2003_Appl.Environ.Microbiol_69_2349
PubMedID: 12676719

Title : Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis - Bohac_2002_Biochemistry_41_14272
Author(s) : Bohac M , Nagata Y , Prokop Z , Prokop M , Monincova M , Tsuda M , Koca J , Damborsky J
Ref : Biochemistry , 41 :14272 , 2002
Abstract : Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds, resulting in the formation of an alcohol, a halide, and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole, and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of S(N)2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB, and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr, and Phe169Leu mutants of LinB were constructed, purified, and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule, and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223, and Val226 of DhlA; Trp207, Pro208, and Ile211 of LinB; and Phe205, Pro206, and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.
ESTHER : Bohac_2002_Biochemistry_41_14272
PubMedSearch : Bohac_2002_Biochemistry_41_14272
PubMedID: 12450392
Gene_locus related to this paper: sphpi-linb

Title : Characterization of a class II defective transposon carrying two haloacetate dehalogenase genes from Delftia acidovorans plasmid pUO1 - Sota_2002_Appl.Environ.Microbiol_68_2307
Author(s) : Sota M , Endo M , Nitta K , Kawasaki H , Tsuda M
Ref : Applied Environmental Microbiology , 68 :2307 , 2002
Abstract : The two haloacetate dehalogenase genes, dehH1 and dehH2, on the 65-kb plasmid pUO1 from Delftia acidovorans strain B were found to be located on transposable elements. The dehH2 gene was carried on an 8.9-kb class I composite transposon (TnHad1) that was flanked by two directly repeated copies of IS1071, IS1071L and IS1071R. The dehH1 gene was also flanked by IS1071L and a truncated version of IS1071 (IS1071N). TnHad1, dehH1, and IS1071N were located on a 15.6-kb class II transposon (TnHad2) whose terminal inverted repeats and res site showed high homology with those of the Tn21-related transposons. TnHad2 was defective in transposition because of its lacking the transposase and resolvase genes. TnHad2 could transpose when the Tn21-encoded transposase and resolvase were supplied in trans. These results demonstrated that Tn Had2 is a defective Tn21-related transposon carrying another class I catabolic transposon.
ESTHER : Sota_2002_Appl.Environ.Microbiol_68_2307
PubMedSearch : Sota_2002_Appl.Environ.Microbiol_68_2307
PubMedID: 11976102
Gene_locus related to this paper: morsp-deh1

Title : A phylogenomic study of the OCTase genes in Pseudomonas syringae pathovars: the horizontal transfer of the argK-tox cluster and the evolutionary history of OCTase genes on their genomes - Sawada_2002_J.Mol.Evol_54_437
Author(s) : Sawada H , Kanaya S , Tsuda M , Suzuki F , Azegami K , Saitou N
Ref : Journal of Molecular Evolution , 54 :437 , 2002
Abstract : Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity. Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al. (1999) showed that the ancestor of P. syringae had diverged into at least three monophyletic groups during its evolution. Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome. The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P. syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements. Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK-tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P. syringae pathovars (pv. actinidiae and pv. phaseolicola) from bacterial species distantly related to P. syringae and that its acquisition was quite recent (i.e., after the ancestor of P. syringae diverged into the respective pathovars). Furthermore, the results of a detailed analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene], which is present within the argK-tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P. aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P. syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK-tox cluster has horizontally transferred onto the genomes of pv. actinidiae and pv. phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars. Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P. syringae.
ESTHER : Sawada_2002_J.Mol.Evol_54_437
PubMedSearch : Sawada_2002_J.Mol.Evol_54_437
PubMedID: 11956683
Gene_locus related to this paper: psesy-PSPTO4540

Title : Naphthalene degrading genes on plasmid NAH7 are on a defective transposon - Tsuda_1990_Mol.Gen.Genet_223_33
Author(s) : Tsuda M , Iino T
Ref : Molecular & General Genetics , 223 :33 , 1990
Abstract : A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class II transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.
ESTHER : Tsuda_1990_Mol.Gen.Genet_223_33
PubMedSearch : Tsuda_1990_Mol.Gen.Genet_223_33
PubMedID: 2175388
Gene_locus related to this paper: psepu-q1xgk7