Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Infection and Immunity
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
MOLECULAR AND CELLULAR PATHOGENESIS

Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coliO157:H7 Deficient in Adherence to Caco-2 Cells

Ichiro Tatsuno, Hiroshi Kimura, Akiko Okutani, Kyoko Kanamaru, Hiroyuki Abe, Shinya Nagai, Kozo Makino, Hideo Shinagawa, Mitsutaka Yoshida, Katsuhiro Sato, Jyunichi Nakamoto, Toru Tobe, Chihiro Sasakawa
Ichiro Tatsuno
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hiroshi Kimura
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Akiko Okutani
Laboratory of Veterinary Public Health, Graduate School of Agriculture and Life Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657,
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kyoko Kanamaru
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hiroyuki Abe
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shinya Nagai
Nippon Institute for Biological Science, 9-2221-1 Shinmachi, Ome, Tokyo 198-0024,
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kozo Makino
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hideo Shinagawa
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mitsutaka Yoshida
Central Laboratory of Medical Science, Division of Electron Microscopy, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Katsuhiro Sato
Central Laboratory of Medical Science, Division of Electron Microscopy, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jyunichi Nakamoto
Central Laboratory of Medical Science, Division of Electron Microscopy, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Toru Tobe
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Chihiro Sasakawa
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/IAI.68.10.5943-5952.2000
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Fig. 1.
    • Open in new tab
    • Download powerpoint
    Fig. 1.

    Adherence phenotypes of class 2 mutants and class 3 mutants are shown. The C8-A2 and C6-H10 mutants are representatives of class 2 mutants. The G1-E11 mutant shows a diffused adherence phenotype. The bacteria grown in DMEM-glycerol for 2 h were used to infect Caco-2 cell monolayers. The infected monolayers were incubated for 1.5 h and washed five times with PBS. After another 2.5 h of incubation, the monolayers were again washed three times, fixed with methanol, and stained with Giemsa solution to visualize the adherent bacterial colonies. WT, wild type.

  • Fig. 2.
    • Open in new tab
    • Download powerpoint
    Fig. 2.

    Genetic organization of the EHEC O157:H7 LEE adapted from Perna et al. (38). Insertion sites of all class 1 mutants and the class 3 mutant (G1-E11) are shown. Genes with a mini-Tn5Km2 insertion are shown by black arrows. Operons LEE1 to LEE4 were those of the EPEC LEE (30).

  • Fig. 3.
    • Open in new tab
    • Download powerpoint
    Fig. 3.

    Levels of secretion and expression of EspA and intimin of class 1 to 3 mutants. Trichloroacetic acid-precipitated culture supernatants (sup) and bacterial cell lysates (whole) derived from equal amounts of wild-type strain O157T, class 1 mutants (A), class 3 mutants (B), and class 2 mutants (C) were resolved by SDS-12% PAGE, transferred to nitrocellulose membrane, and probed with polyclonal rabbit antisera specific to each protein indicated at the right of the photos.

  • Fig. 4.
    • Open in new tab
    • Download powerpoint
    Fig. 4.

    Adherence behavior of O157Sakai (WT), the intimin mutant (Δeae), type III secretion mutant (B9-F9), and EPEC B171-8 (EPEC). B9-F9 is representative of class 1 mutants defective in the type III secretion system. (A) The bacteria were grown at 37°C for 2 h in DMEM-glycerol and then used to infect Caco-2 cell monolayers. Then these infected monolayers were incubated for 1.5 h and washed five times with PBS. After another 0, 1, 2, or 3 h of incubation at 37°C in DMEM-glycerol, the monolayers were again washed three times with PBS, fixed with methanol, and stained with Giemsa solution to visualize the adherent bacterial colonies. Strains and incubation times are shown above and on the left side of photos, respectively. (B) The black bars represent the total number of sites containing adherent EHEC, having either a single bacterium or a cluster of multiple bacteria. The white bars represent the number of clusters containing at least eight bacteria. The data shown are the means and standard errors of the means for 20 microscopic fields. The representative results were obtained from three independent experiments.

  • Fig. 5.
    • Open in new tab
    • Download powerpoint
    Fig. 5.

    Photomicrographs of the intimin mutant and parental wild-type strain in a rhodamine-phalloidin assay for FAS in Caco-2 cells. (A) Caco-2 cells infected by bacteria as described in the Fig. 4legend are shown as follows: in fluorescent views after treatment with anti-O157 LPS rabbit antibody followed by anti-rabbit goat antibody conjugated with fluorescein isothiocyanate (Bacteria), in fluorescent views of actin stained by rhodamine-phalloidin (Actin), and in a superimposed view of bacteria (green) and actin (red) (Super impose). Strains and incubation times are indicated at the left of photos. (B) Quantitative FAS assay of the O157Sakai wild type (WT) and the intimin mutant (Δeae), derived from the experiments whose results are shown in panel A.

Tables

  • Figures
  • Table 1.

    Adherence and other characteristics of strains

    StrainsAdherenceaEspA secretionbIntiminbFAScGrowthdMCe
    WTf+++++++N+
    Class 1
     A3-C10−−+NDN−
     A3-E9−−+NDN−
     A5-E4−−+NDN−
     A6-E7−−+NDN−
     B4-B2−−+NDN−
     B6-H7−−+NDN−
     B8-E7−−+NDN−
     B8-F12−−+NDN−
     B9-F9−−+NDN−
     D5-B12−−+NDN−
    Class 2
     A3-H2++/−++N+
     C6-E9+++++N+
     C6-F9++++N+
     C6-H10+++++N+
     C7-A10++++N+
     C8-A2++++N+
     F4-H4++++N+
     F5-F4++++N+
     F6-B9+++++N+
     F6-D11+++++N+
     G2-B12+++++N+
     G4-B6++++N+
     G4-B12++++N+
     G4-C10+++++N+
     G4-E10++++N+
     H7-C4++++N+
    Class 3
     G1-E11++−−N−
    • ↵a Adherence was divided by the mean adherence (100%) of the O157Sakai-T parental strain. ++++, >80%; +++, 80 to 50%; ++, 50 to 25%; +, 25 to 1%; −, <1%. Data are the means of triplicate determinations, which differed by <15%, from representative experiments.

    • ↵b EspA in culture supernatant and intimin in whole bacterial cell were detected by anti-EspA and anti-intimin antibody, respectively. +, detected; −, not detected; +/−, detected at a reduced level.

    • ↵c FAS assays of infected Caco-2 cells were performed by using rhodamine-phalloidin. ND, not determined because nonadherent to Caco-2 cells.

    • ↵d Growth rates of each mutant and the parental wild type were essentially the same. N, normal.

    • ↵e Ability to form microcolonies; +, positive; −, negative.

    • ↵f WT, wild type.

  • Table 2.

    Products encoded by the gene withaor in the vicinity of mini-Tn5Km2

    StrainGene productSite (min) onE. coli K-12 genomebAccession no. or reference
    A3-H2Hypothetical 17.8-kDa protein (yfjG)59.2P52121 (1)
    C6-E9Putative membrane protein–AE000234
    C6-F9Putative membrane protein–AE000234
    C6-H10Putative membrane protein–AE000234
    C7-A10Putative membrane protein–AE000234
    C8-A2Hypothetical 8-membrane-spanning 37.6-kDa protein (yiaH)80.2P37669
    F4-H4Putative NAGC-like transcriptional regulator (yphHI)57.8AE000341
    ABC transporter-periplasmic binding protein YPHF precursorP77269
    Hypothetical 127.3-kDa proteinP76585
    Serine hydroxy transferase (glyA)38
    F5-F4Putative potassium channel (kch)28.3P31069 (32)
    F6-B9Hypothetical 10-membrane-spanning 58.4-kDa protein27.2P40877
    F6-D11Hypothetical 40.2-kDa protein64.7Q46803
    Hypothetical sigma 54-dependent transcriptional regulatorQ46802
    G2-B12Putative adherence factor (efa1)–AF159462 (34)
    G4-B6Arsenite (efflux) pump membrane protein (arsF)78.597
    Origopeptidase A (prlC)3
    G4-B12Hypothetical 27.7-kDa protein14.6P77627
    G4-C10(orf3 of EPEC pathogenicity island LIM)–46
    G4-E10Lysine-specific permease (lysP)48.543
    Hypothetical transcriptional regulatorP32484
    H7-C4Hypothetical 10-membrane-spanning 43.6-kDa protein87.5P32135
    • ↵a Products encoded by the gene with mini-Tn5Km2 are in bold type.

    • ↵b The insertion sites placed in the DNA sequences specific to the O157Sakai genome sequence are indicated by a dash (–). The sites of C6-E9, C6-F9, C6-H10, and C7-A10 were in the same ∼3-kb regions encoding an open reading frame partially homologous to the b1372 gene, which encodes a putative membrane protein of 1,122 amino acids of E. coli K-12. F6-D11 was placed in a region containing genes encoding a hypothetical sigma 54-dependent transcriptional regulator and a hypothetical 40.2-kDa protein homologous to ornithine carbamoyltransferase. G4-C10 was placed in orf3 in a region homologous to the EPEC pathogenicity island named LIM, encoding a chaperon-like protein involved in the stable expression of BfpA and intimin (47).

PreviousNext
Back to top
Download PDF
Citation Tools
Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coliO157:H7 Deficient in Adherence to Caco-2 Cells
Ichiro Tatsuno, Hiroshi Kimura, Akiko Okutani, Kyoko Kanamaru, Hiroyuki Abe, Shinya Nagai, Kozo Makino, Hideo Shinagawa, Mitsutaka Yoshida, Katsuhiro Sato, Jyunichi Nakamoto, Toru Tobe, Chihiro Sasakawa
Infection and Immunity Oct 2000, 68 (10) 5943-5952; DOI: 10.1128/IAI.68.10.5943-5952.2000

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Infection and Immunity article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coliO157:H7 Deficient in Adherence to Caco-2 Cells
(Your Name) has forwarded a page to you from Infection and Immunity
(Your Name) thought you would be interested in this article in Infection and Immunity.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Enterohemorrhagic Escherichia coliO157:H7 Deficient in Adherence to Caco-2 Cells
Ichiro Tatsuno, Hiroshi Kimura, Akiko Okutani, Kyoko Kanamaru, Hiroyuki Abe, Shinya Nagai, Kozo Makino, Hideo Shinagawa, Mitsutaka Yoshida, Katsuhiro Sato, Jyunichi Nakamoto, Toru Tobe, Chihiro Sasakawa
Infection and Immunity Oct 2000, 68 (10) 5943-5952; DOI: 10.1128/IAI.68.10.5943-5952.2000
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • Notes
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Adhesins, Bacterial
Bacterial Adhesion
Carrier Proteins
DNA Transposable Elements
Escherichia coli O157
Escherichia coli Proteins

Related Articles

Cited By...

About

  • About IAI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #IAIjournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0019-9567; Online ISSN: 1098-5522