Development of host-vector systems for bioremediation using recombinant microorganism

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 15380221
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Boundary agriculture
• Research Institution: Kobe University
• Principal Investigator: MURAKAMI Syuichiro Kobe University, Fac, Agriculture, Dep.Biofunctional chemistry, Associate Professor, 農学部, 助教授 (00243329)
• Project Period (FY): 2003 – 2005
• Keywords: cold shock protein / Rhodococcus / SfiI / ccdB / catechol 1,2-dioxygenase / muconate cycloisomerase / muconolactone isomerase / constitutive gene expression

Research Abstract

Genes encoding catechol 1,2-dioxygenase, cis, cis-muconate cycloisomerase, and muconolactone isomerase, which were constitutively synthesized and involved in the aniline metabolism of Rhodococcus sp. AN-22, were cloned. Primer extension analysis revealed a promoter region which transcribed these genes. Two genes, cspA and capB2, encoding cold shock proteins, were also cloned from this strain, and their promoter regions containing a cspB1 gene were identified.
The transformation method of Rhodococcus sp. AN-22 was improved, and the transformation efficiency increased 15-fold compared with the previous procedures. Lethal genes encoding a transcriptional factor GATA-1 (lethal peptide) or an inhibitor against DNA gyrase were introduced downstream of the cspB1 promoter region, and lethal cassettes activated by a cold condition were constructed. The cassettes were introduced into Rhodococcus sp. AN-22, and the growth of transformants carrying the cassettes were examined at 15℃. The parent stain Rhodococcus sp. AN-22 showed good growth at 15℃; on the other hand, the transformants carrying the cassettes couldn't be grown at 15℃. However, when plates where the transformants were inoculated and didn't show growth at 15℃ were incubated at 30℃, the colonies of transformants appeared on the plates. These results show that the stain AN-22 didn't die out by expression of the lethal genes under the control of the cspB1 promoter although the growth of the strain AN-22 was repressed by their translational products.
To construct a strong lethal cassette, cloning a gene encoding the restriction endonuclease SfiI was attempted. When cloned Sill genes by PCR were sequenced, all genes had mutation, which led translation of the enzyme SfiI to flame shift. These observations show that transformants carrying an intact SfiI gene died out by the expression of the cloned gene.

Research Products

• [Journal Article] Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus sp. AN-22 : production, purification, characterization and gene analysis of CatA, CatB and CatC. (2006)
  • Author(s): Matsumura, E., Sakai, M., Hayashi, K., Murakami, S., Takenaka, S., Aoki, K.
  • Journal Title: Biochem. J. 393 Pages: 219-226
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus sp. AN-22 : production, purification, characterization and gene analysis of CatA, CatB and CatC. (2006)
  • Author(s): Matsumura, E., Sakai, M., Hayashi, K., Murakami, S., Takenaka, S., Aoki, K.
  • Journal Title: Biochem.J. 393(1) Pages: 219-226
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Rhodococcus sp. AN-22のコールドショックタンパク質をコードするcspB2遺伝子のクローニングとプロモーター領域の解析 (2005)
  • Author(s): 村上周一郎, 竹中慎治, 青木健次
  • Journal Title: 日本農芸化学会大会講演要旨集(2005・札幌) Pages: 226
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] アニリン資化性菌Rhodococcus sp. AN-22のコールドショックタンパク質をコードする遺伝子のクローニング (2004)
  • Author(s): 村上周一郎, 竹中慎治, 青木健次
  • Journal Title: 日本農芸化学会大会講演要旨集(2004・広島) Pages: 160
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Constitutive synthesis, purification, and characterization of catechol 1,2-dioxygenase from the aniline-assimilating bacterium Rhodococcussp. AN-22. (2004)
  • Author(s): Matsumura, E., Ooi, S., Murakami, S., Takenaka, S., Aoki, K.
  • Journal Title: Journal of Bioscience and Bioengineering 98,2 Pages: 71-96
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Constitutive synthesis, purification, and characterization of catechol 1,2-dioxygenase from the aniline-assimilating bacterium Rhodococcus sp. AN-22. (2004)
  • Author(s): Matsumura, E., Ooi, S., Murakami, S., Takenaka, S., Aoki, K.
  • Journal Title: Journal of Bioscience and Bioengineering 98,(2) Pages: 71-96
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Rhodococcus sp. AN-22におけるアニリンジオキシゲナーゼ遺伝子の発現特性の解析 (2003)
  • Author(s): 松村栄太郎, 村上周一郎, 竹中慎治, 青木健次
  • Journal Title: 日本農芸化学会大会講演要旨集(2003・藤沢) Pages: 32
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Characterization of the expression of aniline dioxygenase genes from Rhodococcus sp. AN-22.
  • Author(s): Matsumura, E., S., Murakami, S., Takenaka, S., Aoki, K.
  • Journal Title: Annual Meeting of Japan Society of Bioscience, Biotechnology, and Agrochemistry 2003, Fujisawa, abs. Pages: 32
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Cloning of genes encoding cold shock proteins from the aniline-assimilating bacterium Rhodococcus sp. AN-22
  • Author(s): Murakami, S., Takenaka, S., Aoki, K.
  • Journal Title: Annual Meeting of Japan Society of Bioscience Biotechnology, and Agrochemistry 2004, Higashihiroshima, abs. Pages: 160
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Cloning of csp B2 encoding a cold shock protein from Rhodococcus sp. AN-22 and analyses of their promoter regions
  • Author(s): Murakami, S., Takenaka, S., Aoki, K.
  • Journal Title: Annual Meeting of Japan Society of Bioscience, Biotechnology, and Agrochemistry 2005, Sapporo, abs. Pages: 226
  • Description: 「研究成果報告書概要(欧文)」より