Yoshiyuki Ohtsubo

Publications of Yoshiyuki Ohtsubo

• Suppression of pleiotrophic phenotypes of Burkholderia multivorans fur mutant by oxyR mutation.

  Authors: Akane Kimura, Satoshi Yuhara, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

  Microbiology (Reading, England). 02/2012;

  Fur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes suchFur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes such as an inability to efficiently use several carbon sources as well as high sensitivity to hydrogen peroxide (H2O2), paraquat (a superoxide-producing compound), and nitric oxide (NO). To gain more insight into the pleiotropic role of the Fur protein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 fur mutant that restored tolerance to NO were isolated and characterized in this study. The microarray-based comparative genomic analysis and subsequent sequencing analysis indicated that such suppressor mutants had a two-base deletion in the oxyR gene, whose orthologs encode H2O2 -responsive transcriptional regulators in other bacterial species. The suppressor mutants and the reconstructed fur-oxyR double-deletion mutant showed indistinguishable phenotypes in that they were all (i) more resistant than the fur mutant to H2O2, superoxide, NO, and streptonigrin (an iron-activated antibiotic), and (ii) able to use carbon sources that cannot efficiently support the growth of the fur mutant. These results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant. The fur-oxyR double mutants were found to overexpress the KatG (catalase/peroxidase) and AhpC1 and AhpD (alkyl hydroperoxide reductase subunits C and D) proteins, and their enzymatic activities to remove reactive oxygen and nitrogen species were suggested to be responsible for the suppression of phenotypes caused by the fur mutation.

• Pivotal role of anthranilate dioxygenase genes in the adaptation of Burkholderia multivorans ATCC 17616 in soil.

  Authors: Eri Nishiyama, Yoshiyuki Ohtsubo, Yasuhiro Yamamoto, Yuji Nagata, Masataka Tsuda

  FEMS microbiology letters. 02/2012; 330(1):46-55.

  In our recent screen for soil-induced genes, the expression of andA operon (andAcAdAbAa) for anthranilate catabolism in Burkholderia multivorans ATCC 17616 was found to increase dramatically in aIn our recent screen for soil-induced genes, the expression of andA operon (andAcAdAbAa) for anthranilate catabolism in Burkholderia multivorans ATCC 17616 was found to increase dramatically in a soil sample (Nishiyama et al., Environ Microbiol 12: 2539, 2010). The operon was preceded by andR encoding a putative transcriptional regulator for the andA operon. In this study, the andA promoter was induced by tryptophan and anthranilate in an andR-dependent manner. The andA promoter in a deletion mutant lacking tryptophan dioxygenase (one of enzymes for the catabolism of tryptophan to anthranilate) did not respond to tryptophan, indicating that not tryptophan but anthranilate is the effector of AndR. Although both anthranilate and tryptophan were under the detection levels in the soil sample, andA promoter showed higher activity in the soil sample than in a laboratory medium. Such induction required andR and was moderately dependent on the ferric uptake regulator (Fur). The proliferation ability of andAc mutant in the sterile soil was low compared with the co-incubated wild-type cells. These findings suggested that in the soil environment, anthranilate dioxygenase genes are induced by AndR and Fur, and play a pivotal role in the proliferation in the soil environment.

• Genomic organization and genomic structural rearrangements of Sphingobium japonicum UT26, an archetypal γ-hexachlorocyclohexane-degrading bacterium.

  Authors: Yuji Nagata, Shunsuke Natsui, Ryo Endo, Yoshiyuki Ohtsubo, Natsuko Ichikawa, Akiho Ankai, Akio Oguchi, Shigehiro Fukui, Nobuyuki Fujita, Masataka Tsuda

  Enzyme and microbial technology. 12/2011; 49(6-7):499-508.

  The complete genome sequencing of a γ-hexachlorocyclohexane-degrading strain, Sphingobium japonicum UT26, revealed that the genome consists of two circular chromosomes [with sizes of 3.5 Mb (Chr1)The complete genome sequencing of a γ-hexachlorocyclohexane-degrading strain, Sphingobium japonicum UT26, revealed that the genome consists of two circular chromosomes [with sizes of 3.5 Mb (Chr1) and 682kb (Chr2)], a 191-kb large plasmid (pCHQ1), and two small plasmids with sizes of 32 and 5kb. The lin genes are dispersed on Chr1, Chr2, and pCHQ1. Comparison of the UT26 genome with those of other sphingomonad strains demonstrated that the "specific"lin genes for conversion of γ-HCH to β-ketoadipate (linA, linB, linC, linRED, and linF) are located on the DNA regions unique to the UT26 genome, suggesting the acquisition of these lin genes by horizontal transfer events. On the other hand, linGHIJ and linKLMN are located on the regions conserved in the genomes of sphingomonads, suggesting that the linGHIJ-encoded β-ketoadipate pathway and the LinKLMN-type ABC transporter system are involved in core functions of sphingomonads. Based on these results, we propose a hypothesis that UT26 was created by recruiting the specific lin genes into a strain having core functions of sphingomonads. Most of the specific lin genes in UT26 are associated with IS6100. Our analysis of spontaneous linA-, linC-, and linRED-deletion mutants of UT26 revealed the involvement of IS6100 in their deduced genome rearrangements. These facts strongly suggest that IS6100 plays important roles both in the dissemination of the specific lin genes and in the genome rearrangements.

• The lin genes for γ-hexachlorocyclohexane degradation in Sphingomonas sp. MM-1 proved to be dispersed across multiple plasmids.

  Authors: Michiro Tabata, Ryo Endo, Michihiro Ito, Yoshiyuki Ohtsubo, Ashwani Kumar, Masataka Tsuda, Yuji Nagata

  Bioscience, biotechnology, and biochemistry. 03/2011; 75(3):466-72.

  A γ-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of γ-HCH led to the detection ofA γ-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of γ-HCH led to the detection of five γ-HCH metabolites, γ-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 γ-HCH degradation originally identified in the well-studied γ-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 γ-HCH to β-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.

• Complete genome sequence of the representative γ-hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26.

  Authors: Yuji Nagata, Yoshiyuki Ohtsubo, Ryo Endo, Natsuko Ichikawa, Akiho Ankai, Akio Oguchi, Shigehiro Fukui, Nobuyuki Fujita, Masataka Tsuda

  Journal of bacteriology. 11/2010; 192(21):5852-3.

  Sphingobium japonicum strain UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as aSphingobium japonicum strain UT26 utilizes γ-hexachlorocyclohexane (γ-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 γ-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.

• Identification of Burkholderia multivorans ATCC 17616 genes induced in soil environment by in vivo expression technology.

  Authors: Eri Nishiyama, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

  Environmental microbiology. 04/2010; 12(9):2539-58.

  Burkholderia multivorans ATCC 17616 was originally isolated from a soil sample, and it carries three chromosomes. To identify traits of likely adaptive significance for colonization of soil, an inBurkholderia multivorans ATCC 17616 was originally isolated from a soil sample, and it carries three chromosomes. To identify traits of likely adaptive significance for colonization of soil, an in vivo expression technology system for ATCC 17616 was constructed using the promoterless and tandemly arranged dapB and lacZ genes as the reporters, and this system was applied to identify the genomic loci of ATCC 17616 that were induced in sterilized soil. Our screening of a library consisting of dapB-lacZ-inserted clones resulted in the isolation of 713 clones in which the insertion sites of genome were putatively transcribed in the soil but not in laboratory media. All insertion sites in the genome were determined by high-throughput sequencing using genomic DNA as the templates, and subsequent analysis led to a reliable list of a total of 116 genomic loci as the B. multivorans ATCC 17616 loci induced in a soil environment (mls). These 116 mls carried the genes for energy acquisition from various substances, as well as genes for cell-envelope integrity and the niche adaptation. The distribution of these loci was biased to the second chromosome, suggesting the importance of this replicon as a source of adaptive traits enhancing survival of this organism in natural environments.

• Analysis of extracellular alginate lyase and its gene from a marine bacterial strain, Pseudoalteromonas atlantica AR06.

  Authors: Ryoji Matsushima, Hiroko Danno, Motoharu Uchida, Kenji Ishihara, Toshiyuki Suzuki, Masaki Kaneniwa, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

  Applied microbiology and biotechnology. 10/2009;

  Pseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivationPseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivation media exhibited alginate lyase activity to depolymerize the alginate molecules having homopolymeric and heteropolymeric forms of mannuronate and guluronate so as to mainly convert into the dimer to tetramer. A DNA fragment encoding a portion of alginate lyase was amplified from AR06 genomic DNA by PCR using a set of degenerated primers, and then the whole alginate lyase gene, named alyA, and its flanking regions were obtained from a cosmid library of AR06 genomic DNA. The alyA mutant of AR06 showed (1) the loss of alginate depolymerization activity on alginate agar plate and (2) significant growth defects in alginate minimal medium; these defects were complemented by the introduction of the alyA gene. Furthermore, zymography and biochemical analyses revealed that three extracellular protein bands of AR06 had alginate lyase activities and that all three protein bands were derived from the nascent alyA gene product. These results clearly indicated that the alyA gene greatly contributes to the assimilation of alginate in AR06. The transcription of the alyA gene was induced by the presence of alginate in minimal medium, but its obvious induction was not observed in rich medium even in the presence of alginate.

• Novel organization of aromatic degradation pathway genes in a microbial community as revealed by metagenomic analysis.

  Authors: Hikaru Suenaga, Yoshinori Koyama, Masatoshi Miyakoshi, Ryo Miyazaki, Hirokazu Yano, Masahiro Sota, Yoshiyuki Ohtsubo, Masataka Tsuda, Kentaro Miyazaki

  The ISME journal. 08/2009;

  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,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.The ISME Journal advance online publication, 9 July 2009; doi:10.1038/ismej.2009.76.

• GenomeMatcher: A graphical user interface for DNA sequence comparison.

  Authors: Yoshiyuki Ohtsubo, Wakako Ikeda-Ohtsubo, Yuji Nagata, Masataka Tsuda

  BMC bioinformatics. 10/2008; 9(1):376.

  ABSTRACT: BACKGROUND: The number of available genome sequences is increasing, and easy-to-use software that enables efficient comparative analysis is needed. RESULTS: We developed GenomeMatcher, aABSTRACT: BACKGROUND: The number of available genome sequences is increasing, and easy-to-use software that enables efficient comparative analysis is needed. RESULTS: We developed GenomeMatcher, a stand-alone software package for Mac OS X. GenomeMatcher executes BLAST and MUMmer, and the detected similarities are displayed in two-dimensional and parallel views with similarity values indicated by color. Selection and re-computation of any subregions is easily performed and allows flexible and in-depth analysis. Furthermore, symbols for annotation data are displayed along the views, and the user can relate the genomic differences with annotation data. While bl2seq allows sub-Giga base comparison, three alignment programs, bl2seq, MAFFT and ClustalW, together with a dotmatch program allow comparative analysis of single- nucleotide level resolution. GenomeMatcher images can be saved as PDF and TIFF files for presentation. As examples of graphical ability of GenomeMatcher to show similarity in colors, we show two cases in Burkholderia and Vivrio strains that the nucleotide sequence of the second largest chromosome changes more rapidly than the largest chromosome. CONCLUSIONS: GenomeMatcher is efficient and easy-to-use stand-alone software for in-depth comparative analysis of two sequences. GenomeMatcher is useful for detecting similarities in DNA sequences ranging in size from a few to sub-Giga bases.

• Construction of signature-tagged mutant library in Mesorhizobium loti as a powerful tool for functional genomics.

  Authors: Yoshikazu Shimoda, Hisayuki Mitsui, Hiroko Kamimatsuse, Kiwamu Minamisawa, Eri Nishiyama, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda, Sayaka Shinpo, Akiko Watanabe, Mitsuyo Kohara, Manabu Yamada, Yasukazu Nakamura, Satoshi Tabata, Shusei Sato

  DNA research : an international journal for rapid publication of reports on genes and genomes. 10/2008; 15(5):297-308.

  Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wideRhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29,330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.

• Characterization of the traD Operon of Naphthalene-Catabolic Plasmid NAH7: a Host-Range Modifier in Conjugative Transfer.

  Authors: Ryo Miyazaki, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

  Journal of bacteriology. 09/2008;

  Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transferPseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are situated three genes, traD, traE, and traF, whose functions and roles in conjugation had remained unclear. These three genes were transcribed monocistronically and thus termed the traD operon. The mutations of the three genes in the traD operon resulted in 10- to 10(5)-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas/Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in the G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer: TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating-pair formation system).

• Insertion sequence-based cassette PCR: cultivation-independent isolation of gamma-hexachlorocyclohexane-degrading genes from soil DNA.

  Authors: Genki Fuchu, Yoshiyuki Ohtsubo, Michihiro Ito, Ryo Miyazaki, Akira Ono, Yuji Nagata, Masataka Tsuda

  Applied microbiology and biotechnology. 07/2008; 79(4):627-32.

  gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100gamma-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.

• Pleiotropic roles of iron-responsive transcriptional regulator Fur in Burkholderia multivorans.

  Authors: Satoshi Yuhara, Harunobu Komatsu, Hiroyuki Goto, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

  Microbiology (Reading, England). 06/2008; 154(Pt 6):1763-74.

  The fur (ferric uptake regulator) gene of Burkholderia multivorans ATCC 17616 was identified by transposon mutagenesis analysis. The fur deletion mutant of strain ATCC 17616 (i) constitutivelyThe fur (ferric uptake regulator) gene of Burkholderia multivorans ATCC 17616 was identified by transposon mutagenesis analysis. The fur deletion mutant of strain ATCC 17616 (i) constitutively produced siderophores, (ii) was more sensitive to reactive oxygen species (ROS) than the wild-type strain, (iii) showed lower superoxide dismutase and catalase activities than the wild-type strain, (iv) was unable to grow on M9 minimal agar plates containing several substrates that can be used as sole carbon sources by the wild-type strain, and (v) was hypersensitive to nitrite and nitric oxide under microaerobic and aerobic conditions, respectively. These results clearly indicate that the Fur protein in strain ATCC 17616 plays pleiotropic roles in iron homeostasis, removal and/or resistance to ROS and nitrosative stress, and energy metabolism. Furthermore, employment of an in vivo Fur titration assay system led to the isolation from the ATCC 17616 genome of 13 Fur-binding DNA regions, and a subsequent electrophoretic mobility-shift assay confirmed the direct binding of Fur protein to all of these DNA regions. Transcriptional analysis of the genes located just downstream of the Fur-binding sites demonstrated that Fur acts as a repressor for these genes. Nine of the 13 regions were presumed to be involved in the acquisition and utilization of iron.

• Aerobic degradation of lindane (gamma-hexachlorocyclohexane) in bacteria and its biochemical and molecular basis.

  Authors: Yuji Nagata, Ryo Endo, Michihiro Ito, Yoshiyuki Ohtsubo, Masataka Tsuda

  Applied microbiology and biotechnology. 10/2007; 76(4):741-52.

  gamma-Hexachlorocyclohexane (gamma-HCH, also called gamma-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway ofgamma-Hexachlorocyclohexane (gamma-HCH, also called gamma-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of gamma-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. gamma-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the gamma-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the gamma-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.

• 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.

  Authors: Hirokazu Yano, Christine E Garruto, Masahiro Sota, Yoshiyuki Ohtsubo, Yuji Nagata, Gerben J Zylstra, Peter A Williams, Masataka Tsuda

  Journal of molecular biology. 06/2007; 369(1):11-26.

  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 elucidateRecent 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.

• Identification and characterization of genes encoding a putative ABC-type transporter essential for utilization of gamma-hexachlorocyclohexane in Sphingobium japonicum UT26.

  Authors: Ryo Endo, Yoshiyuki Ohtsubo, Masataka Tsuda, Yuji Nagata

  Journal of bacteriology. 06/2007; 189(10):3712-20.

  Sphingobium japonicum UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes forSphingobium japonicum UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes for the conversion of gamma-HCH to beta-ketoadipate in UT26. In this study, we analyzed a mutant obtained by transposon mutagenesis and identified and characterized new genes encoding a putative ABC-type transporter essential for the utilization of gamma-HCH in strain UT26. This putative ABC transporter consists of four components, permease, ATPase, periplasmic protein, and lipoprotein, encoded by linK, linL, linM, and linN, respectively. Mutation and complementation analyses indicated that all the linKLMN genes are required, probably as a set, for gamma-HCH utilization in UT26. Furthermore, the mutant cells deficient in this putative ABC transporter showed (i) higher gamma-HCH degradation activity and greater accumulation of the toxic dead-end product 2,5-dichlorophenol (2,5-DCP), (ii) higher sensitivity to 2,5-DCP itself, and (iii) higher permeability of hydrophobic compounds than the wild-type cells. These results strongly suggested that LinKLMN are involved in gamma-HCH utilization by controlling membrane hydrophobicity. This study clearly demonstrated that a cellular factor besides catabolic enzymes and transcriptional regulators is essential for utilization of xenobiotic compounds in bacterial cells.

• Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches.

  Authors: Akira Ono, Ryo Miyazaki, Masahiro Sota, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

  Applied microbiology and biotechnology. 03/2007; 74(2):501-10.

  Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of aTwo different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway.

• Complete nucleotide sequence of an exogenously isolated plasmid, pLB1, involved in gamma-hexachlorocyclohexane degradation.

  Authors: Ryo Miyazaki, Yukari Sato, Michihiro Ito, Yoshiyuki Ohtsubo, Yuji Nagata, Masataka Tsuda

  Applied and environmental microbiology. 12/2006; 72(11):6923-33.

  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 haloalkaneThe 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.

• Identification of a response regulator gene for catabolite control from a PCB-degrading beta-proteobacteria, Acidovorax sp. KKS102.

  Authors: Yoshiyuki Ohtsubo, Hiroyuki Goto, Yuji Nagata, Toshiaki Kudo, Masataka Tsuda

  Molecular microbiology. 07/2006; 60(6):1563-75.

  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 underAcidovorax 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.

• Growth inhibition by metabolites of gamma-hexachlorocyclohexane in Sphingobium japonicum UT26.

  Authors: Ryo Endo, Yoshiyuki Ohtsubo, Masataka Tsuda, Yuji Nagata

  Bioscience, biotechnology, and biochemistry. 05/2006; 70(4):1029-32.

  The growth of a gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacterium Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 in rich medium was inhibited by gamma-HCH. This growthThe growth of a gamma-hexachlorocyclohexane (gamma-HCH)-degrading bacterium Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 in rich medium was inhibited by gamma-HCH. This growth inhibition was not observed in a mutant that lacked the initial or second step enzymatic activity for gamma-HCH degradation, suggesting that metabolites of gamma-HCH are toxic to UT26. Two metabolites of gamma-HCH, 2,5-dichlorophenol (2,5-DCP) and 2,5-dichlorohydroquinone (2,5-DCHQ), showed a direct toxic effect on UT26 and other sphingomonad strains. Because only 2,5-DCP accumulated during gamma-HCH degradation, 2,5-DCP is thought to be a main compound for growth inhibition.