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Genetic and molecular analysis of bacteriocins of enterococcus clinical isolates-a bacterial colonization factor-

Research Project

Project/Area Number 19390123
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Bacteriology (including Mycology)
Research InstitutionGunma University

Principal Investigator

IKE Yasuyoshi  Gunma University, 大学院・医学系研究科, 教授 (60125820)

Co-Investigator(Kenkyū-buntansha) TOMITA Haruyoshi  群馬大学, 大学院・医学系研究科, 准教授 (70282390)
INOUE Takako  群馬大学, 大学院・医学系研究科, 助教 (00431700)
Co-Investigator(Renkei-kenkyūsha) TANIMOTO Koichi  群馬大学, 大学院・医学系研究科, 准教授 (40188389)
FUJIMOTO Shuhei  群馬大学, 大学院・医学系研究科, 講師 (90241869)
NOMURA Takahiro  群馬大学, 大学院・医学系研究科, 技術専門職員 (30396634)
Project Period (FY) 2007 – 2009
Project Status Completed (Fiscal Year 2009)
Budget Amount *help
¥18,720,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥4,320,000)
Fiscal Year 2009: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
Fiscal Year 2008: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
Fiscal Year 2007: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000)
Keywords病原性 / 腸球菌 / バクテリオシン / バクテリオシン遺伝子 / 分子遺伝 / プラスミド
Research Abstract

1. The conjugative plasmid pYI14 (61kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E.faecalis. Genetic analysis showed that the determinant was located in a 6.6-kbp region Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL_1) ORF8 (bacL_2), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL_1, bacL_2, bacA, and bacI were essential for expression of the bacteriocin in E.faecalis. Extracellular complementation of bacteriocin expression was possible for bacL_1 and -L_2 and bacA mutants. bacL_1 and -L_2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, … More providing the host with resistance to its own bacteriocin activity.
2. The bacteriocin 51 (Bac51) was encoded on the mobilizable plasmid pHY (6,037bp) isolated from vancomycin resistant E.faecium V38. Bacteriocin 51 was active against E.faecium, E.hirae, and E.duraus. The sequence analysis of pHY showed that plasmid pHY encoded nine ORFs from ORF1 to ORF9 in this order. ORF3, ORF4 and ORF5 showed homology to mobC, mobA, and mobB, respectively. ORF7 and ORF8 showed homology to repA and repB, respectively. ORF1, ORF2, ORF6 and ORF9 had no homology to the reported genes. The Tn5 insertion mutants in ORF1 did not show both bacteriocin and immunity activities, implying that ORF1 and ORF2 were the bacteriocin determinant, and were designated as bacA and bacB, respectively. Detailed DNA sequence analysis of bacA and bacB was performed. bacA encoded a 144-amino-acid protein. The ATG start codon was preceded by a potential ribosome binding site 8 bp upstream. The deduced bacA protein had a span of hydrophobic residues typical of the signal sequence at its amino terminus and a potential signal peptidase processing site corresponding to the VEA sequence was located at position 37 to 39 amino acid. The predicted BacA mature protein consisted of 105 amino acids. There was a potential promoter sequence upstream of the start codon. bacB encoded a 55 amino acid protein. The ATG start codon was preceded by a potential ribosome binding site 12 bp upstream. There was no obvious promoter sequence upstream of the ribosome binding site. A putative transcription terminator signal was identified downstream of bacB. There was no obvious promoter or terminator sequence between bacA and bacB. The transcript of bacA and bacB were analyzed by Northern hybridization. The results of Northern analysis of bacA and bacB with bacA-RNA probe showed about 700 nucleotides which corresponded to the nucleotide size of both bacA and bacB, indicating that a transcription initiated from the promoter upstream bacA, continued through bacB, and terminated at the terminator downstream of bacB. The transcription start site was determined at the nucleotide T located at six nucleotides downstream from -10 promoter sequence by Rapid Amplification of cDNA Ends (RACE) method. These results indicated that the bacA and bacB composed of operon structure, and bacA was bacteriocin structure gene and bacB was the immunity gene. Less

Report

(4 results)
  • 2009 Annual Research Report   Final Research Report ( PDF )
  • 2008 Annual Research Report
  • 2007 Annual Research Report
  • Research Products

    (23 results)

All 2010 2009 2008 2007

All Journal Article (14 results) (of which Peer Reviewed: 13 results) Presentation (9 results)

  • [Journal Article] Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections : First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin, and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac)2009

    • Author(s)
      Zheng B., Tomita H., Inoue T., Ike Y.
    • Journal Title

      Antimicrob Agents chemother 53(2)

      Pages: 735-747

    • Related Report
      2009 Final Research Report
    • Peer Reviewed
  • [Journal Article] Nationwide epidemiological study revealed the dissemination of meticillin-resistant Staphylococcus aureus carrying a specific set of virulence-associated genes in Japanese hospitals2009

    • Author(s)
      Ohkura T, Yamada K, Okamoto A, Baba H, Ike Y, Arakawa Y, Hasegawa T, Ohta M
    • Journal Title

      Journal of Medical Microbiology 58

      Pages: 1329-1336

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Assessment of antibiotic resistance in probiotic strain Lactobacillus brevis KB2902009

    • Author(s)
      Fukao M, Tomita H, Yakabe T, Nomura T, Ike Y, Yajima N
    • Journal Title

      Journal of Food Protection 72

      Pages: 1923-1929

    • Related Report
      2009 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections: First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin, and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac).2009

    • Author(s)
      Zheng B., Tomita H., Inoue T. and Ike Y.
    • Journal Title

      Antimicrob Agents Chemother. 53(2)

      Pages: 735-747

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Genetic Analysis of the Enterococcus Vancomycin-Resistance Conjugative Plasmid pHTβ : Identification of the Region involved in Cell Aggregation and traB, a Key Regulator Gene for Plasmid Transfer and Cell Aggregation2008

    • Author(s)
      Tomita H. Ike Y.
    • Journal Title

      J Bacteriol 190(23)

      Pages: 7739-7753

    • Related Report
      2009 Final Research Report
    • Peer Reviewed
  • [Journal Article] Cloning and genetic analyses of the bacteriocin 41 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14 : a novel bacteriocin complemented by two extracellular components (lysin and activator)2008

    • Author(s)
      Tomita H., kamei E., Ike Y.
    • Journal Title

      J Bacteriol 190(6)

      Pages: 2075-85

    • Related Report
      2009 Final Research Report
    • Peer Reviewed
  • [Journal Article] Cloning and genetic analyses of the bacteriocin 41 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14: a novel bacteriocin complemented by two extracellular components (lysin and activator).2008

    • Author(s)
      Tomita H, kamei E, Ike Y.
    • Journal Title

      J Bacteriol 190(6)

      Pages: 2075-2085

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Fluoroquinolone enhances the mutation frequency for meropenem-selected carbapenem resistance in Pseudomonas aeruginosa, but use of the high-potency drug doripenem inhibits mutant formation.2008

    • Author(s)
      Tanimoto K., Tomita H., Fujimoto S., Okuzumi K. and Ike Y.
    • Journal Title

      Antimicrob Agents Chemother. 52(10)

      Pages: 3795-3800

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Complete nucleotide sequencing and analysis of the 65-kb highly conjugative Enterococcus faecium plasmid pMG1: identification of the transfer-related region and the minimum region reauired for replication2008

    • Author(s)
      Tanimoto K., and Ike Y.
    • Journal Title

      FEMS Microbiol Lett. 288

      Pages: 186-195

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Genetic Analysis of the Enterococcus Vancomycin-Resistance Conjugative Plasmid pHTβ: Identification of the Region involved in Cell Aggregation and traB, a Key Regulator Gene for Plasmid Transfer and Cell Aggregation.2008

    • Author(s)
      Tomita H. and Ike Y.
    • Journal Title

      J Bacteriol. 190(23)

      Pages: 7739-7753

    • Related Report
      2008 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cloning and genetic analyses of the bacterdocin 41 determinant encoded on the Enterocoαeus faecalis pheromone-responsive conjugative Plasmid pYI14.2008

    • Author(s)
      Tomita H, Kamei E, and Ike Y
    • Journal Title

      Journal of Bacteriology 190

      Pages: 2075-2085

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Molecular characterization of vancomycin-resistant Enteroooccusfaecium isolates from mainland China2007

    • Author(s)
      Zheng B, Tomita H, Xiao YH, Wang S, Li Y, and Ike Y
    • Journal Title

      Journal of Clinical Microbiology 45

      Pages: 307-312

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] The frrst molecular analysis of clinical isolates of VanA-type vancomycin-resistant Enterocoeeus faθeium strains in Mainland China2007

    • Author(s)
      Zheng B, Tomita H, Xiao YH, and Ike Y
    • Journal Title

      Letters in Applied Microbiology 45

      Pages: 307-312

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] グラム陽性球菌のバンコマイシン耐性獲得と病原性2007

    • Author(s)
      富田 治芳, 池康 嘉
    • Journal Title

      整形・災害外科 50

      Pages: 634-635

    • Related Report
      2007 Annual Research Report
  • [Presentation] 多剤β-ラクタム薬耐性と抗緑膿菌薬2010

    • Author(s)
      池康嘉
    • Organizer
      第83回日本細菌学会総会
    • Place of Presentation
      パシフィコ横浜 (神奈川)
    • Year and Date
      2010-03-27
    • Related Report
      2009 Final Research Report
  • [Presentation] 腸球菌の薬剤耐性と接合伝達性プラスミド2010

    • Author(s)
      谷本弘一
    • Organizer
      第83回日本細菌学会総会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2010-03-27
    • Related Report
      2009 Annual Research Report
  • [Presentation] 多剤β-ラクタム薬耐性と抗緑膿菌薬-高用量ペニシリン系薬の細菌学的考察2010

    • Author(s)
      池康嘉
    • Organizer
      第83回日本細菌学会総会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2010-03-27
    • Related Report
      2009 Annual Research Report
  • [Presentation] 臨床分離腸球菌E.faeciumが生産するバクテリオシン(Bac32)の精製と抗菌作用機序2010

    • Author(s)
      井上貴子
    • Organizer
      第83回日本細菌学会総会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2010-03-27
    • Related Report
      2009 Annual Research Report
  • [Presentation] 院内感染分離VREのvanA型遺伝子のvanY,Zの欠失とテイコプラニン低度耐性について2010

    • Author(s)
      野村隆浩
    • Organizer
      第83回日本細菌学会総会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2010-03-27
    • Related Report
      2009 Annual Research Report
  • [Presentation] 臨床分離VRE E.faeciumが生産する新しいバクテリオシンBac51の遺伝学的解析2010

    • Author(s)
      山下均
    • Organizer
      第83回日本細菌学会総会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2010-03-27
    • Related Report
      2009 Annual Research Report
  • [Presentation] Vancomycin-Resistant Enterococci (VRE) in Japan2010

    • Author(s)
      池康嘉
    • Organizer
      第21回日本臨床微生物学会総会 (日韓合同シンポジウム)
    • Place of Presentation
      東京ドームホテル (東京)
    • Year and Date
      2010-01-31
    • Related Report
      2009 Final Research Report
  • [Presentation] Vancomycin-Resistant Enterococci(VRE)in Japan2010

    • Author(s)
      池康嘉
    • Organizer
      第21回日本臨床微生物学会総会(日韓合同シンポジウム)
    • Place of Presentation
      東京ドームホテル
    • Year and Date
      2010-01-31
    • Related Report
      2009 Annual Research Report
  • [Presentation] Genetic analysis of the Enterocoocus vancomycin-resistance conjugative plasmid pHTβ2007

    • Author(s)
      Tomita H and Ike Y
    • Organizer
      AWaji intemational forum
    • Place of Presentation
      兵庫県・淡路
    • Related Report
      2007 Annual Research Report

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Published: 2007-04-01   Modified: 2016-04-21  

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