Yuji Nagata

Publications of Yuji Nagata

• 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.

• Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature.

  Authors: Radka Chaloupkova, Zbynek Prokop, Yukari Sato, Yuji Nagata, Jiri Damborsky

  The FEBS journal. 06/2011; 278(15):2728-38.

  The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study.The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Δ(R-S)ΔH and Δ(R-S)ΔS were determined. The thermodynamic analysis revealed that the resolution of β-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of α-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase.

• 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.

• Biochemical characteristics of the novel haloalkane dehalogenase DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58.

  Authors: Khomaini Hasan, Andrea Fortova, Tana Koudelakova, Radka Chaloupkova, Mayuko Ishitsuka, Yuji Nagata, Jiri Damborsky, Zbynek Prokop

  Applied and environmental microbiology. 12/2010; 77(5):1881-4.

  We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair ofWe report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.

• 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.

• Crystal structure of γ-hexachlorocyclohexane Dehydrochlorinase LinA from Sphingobium japonicum UT26.

  Authors: Masahiko Okai, Keiko Kubota, Masao Fukuda, Yuji Nagata, Koji Nagata, Masaru Tanokura

  Journal of molecular biology. 10/2010; 403(2):260-9.

  LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from γ hexachlorocyclohexane (HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene via γ-pentachlorocyclohexene. WeLinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from γ hexachlorocyclohexane (HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene via γ-pentachlorocyclohexene. We determined the crystal structure of LinA at 2.25 Å by single anomalous dispersion. LinA exists as a homotrimer, and each protomer forms a cone-shaped α+β barrel fold. The C-terminal region of LinA is extended to the neighboring subunit, unlike that of scytalone dehydratase from Magnaporthe grisea, which is one of the most structurally similar proteins identified by the DALI server. The structure we obtained in this study is in open form, in which γ-HCH can enter the active site. There is a hydrophobic cavity inside the barrel fold, and the active site is largely surrounded by the side chains of K20, L21, V24, D25, W42, L64, F68, C71, H73, V94, L96, I109, F113, and R129. H73 was considered to function as a base that abstracts the proton of γ-HCH through its interaction with D25. Docking simulations with γ-HCH and γ-pentachlorocyclohexene suggest that 11 residues (K20, I44, L64, V94, L96, I109, A111, F113, A131, C132, and T133) are involved in the binding of these compounds and support the degradation mechanism.

• Complete nucleotide sequence of TOL plasmid pDK1 provides evidence for evolutionary history of IncP-7 catabolic plasmids.

  Authors: Hirokazu Yano, Masatoshi Miyakoshi, Kenshiro Ohshima, Michiro Tabata, Yuji Nagata, Masahira Hattori, Masataka Tsuda

  Journal of bacteriology. 09/2010; 192(17):4337-47.

  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 128,921To 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.

• Enantioselectivity of haloalkane dehalogenases and its modulation by surface loop engineering.

  Authors: Zbynek Prokop, Yukari Sato, Jan Brezovsky, Tomas Mozga, Radka Chaloupkova, Tana Koudelakova, Petr Jerabek, Veronika Stepankova, Ryo Natsume, Jan G E van Leeuwen, Dick B Janssen, Jan Florian, Yuji Nagata, Toshiya Senda, Jiri Damborsky

  Angewandte Chemie (International ed. in English). 08/2010; 49(35):6111-5.

• 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.

• Haloalkane dehalogenase from Agrobacterium tumefaciens C58 with new halide-stabilising residues and unique kinetics

  Authors: Khomaini Hasan, Táňa Koudeláková, Andrea Fořtová, Radka Chaloupková, Yuji Nagata, Jiří Damborský, Zbyněk Prokop

  Trends in Enzymology, Ascona, Switzerland; 01/2010

• 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.

• Crystallization and preliminary X-ray analysis of gamma-hexachlorocyclohexane dehydrochlorinase LinA from Sphingobium japonicum UT26.

  Authors: Masahiko Okai, Keiko Kubota, Masao Fukuda, Yuji Nagata, Koji Nagata, Masaru Tanokura

  Acta crystallographica. Section F, Structural biology and crystallization communications. 09/2009; 65(Pt 8):822-4.

  LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from gamma-hexachlorocyclohexane (gamma-HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) viaLinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from gamma-hexachlorocyclohexane (gamma-HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via gamma-pentachlorocyclohexene (gamma-PCCH). LinA was crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals belonged to space group P4(1) or P4(3), with unit-cell parameters a = b = 68.9, c = 101.9 A, and diffracted X-rays to 2.25 A resolution. The crystal contained three molecules in the asymmetric unit.

• Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate.

  Authors: Martina Pavlova, Martin Klvana, Zbynek Prokop, Radka Chaloupkova, Pavel Banas, Michal Otyepka, Rebecca C Wade, Masataka Tsuda, Yuji Nagata, Jiri Damborsky

  Nature chemical biology. 09/2009;

  Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wildEngineering 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.

• Crystallization and preliminary X-ray analysis of a novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94.

  Authors: Tatyana Prudnikova, Tomas Mozga, Pavlina Rezacova, Radka Chaloupkova, Yukari Sato, Yuji Nagata, Jiri Brynda, Michal Kuty, Jiri Damborsky, Ivana Kuta Smatanova

  Acta crystallographica. Section F, Structural biology and crystallization communications. 05/2009; 65(Pt 4):353-6.

  A novel enzyme, DbeA, belonging to the haloalkane dehalogenase family (EC 3.8.1.5) was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to the DbjA enzymeA novel enzyme, DbeA, belonging to the haloalkane dehalogenase family (EC 3.8.1.5) was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to the DbjA enzyme from B. japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. In order to understand the altered activity and specificity of DbeA, its mutant variant DbeA1, which carries the unique fragment of DbjA, was also constructed. Both wild-type DbeA and DbeA1 were crystallized using the sitting-drop vapour-diffusion method. The crystals of DbeA belonged to the primitive orthorhombic space group P2(1)2(1)2(1), while the crystals of DbeA1 belonged to the monoclinic space group C2. Diffraction data were collected to 2.2 A resolution for both DbeA and DbeA1 crystals.

• 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.