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

Expression of Listeriolysin O and ActA by Intracellular and Extracellular Listeria monocytogenes

Marlena A. Moors, Brian Levitt, Philip Youngman, Daniel A. Portnoy
Marlena A. Moors
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Brian Levitt
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Philip Youngman
Department of Genetics, University of Georgia, Athens, Georgia 30602
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Daniel A. Portnoy
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/IAI.67.1.131-139.1999
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

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

    Construction of transcriptional reporter vector pLCR. Plasmids pTV53 and pTV30, which provided the promoterless B. pumilus cat-86 gene and the E. coli lacZ gene with an upstream spoVG ribosome binding site (rbs) and aBamHI site, respectively, were described previously (28, 37). Plasmid pTV1OK (13) provided the kanamycin resistance gene (kan), a type III aminoglycoside phosphotransferase derived from Streptococcus faecalis(35), and the pWVO1ts origin of replication, a temperature-sensitive derivative of the origin of replication fromLactococcus lactis plasmid pWVO1 (18). Plasmid pTV30cat was isolated from B. subtilis by selection for chloramphenicol resistance at 30°C following recombination between plasmids pTV30 and pTV53. Similarly, pTV30catts was isolated by selection for kanamycin resistance at 28°C following recombination between plasmids pTV30cat and pTV1OK. Details of plasmid construction by in vivo recombination in B. subtilis are described in Materials and Methods. pLCR was generated in two conventional cloning steps by partial XbaI digestion and religation of pTV30catts followed by insertion of a PCR-generated fragment containing the trpA terminator (7) into theNcoI and BamHI sites. X, XbaI; E,EcoRI; K, KpnI; P, PstI; N,NcoI; B, BamHI. E, N, and B are unique restriction sites in pLCR.

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

    Schematic of pLCR fusion constructs integrated into theL. monocytogenes chromosome. (A) Integration of pLCR by homologous recombination between the cloned hlypromoter-containing fragment (hly′) and the corresponding region of the L. monocytogenes chromosome, yielding strain DP-L2986. (B and C) Integration structures which resulted from recombination between the actA promoter-containing fragment (actA′) cloned in the forward (B) and reverse (C) orientations with respect to the transcription of lacZ andcat and the corresponding region of the L. monocytogenes chromosome, yielding strains DP-L2989 and DP-L2812, respectively. Broken lines represent plasmid sequences, and solid lines represent flanking chromosomal regions. Arrows depict promoter positions and the direction of transcription for each of the indicated genes. Stem and loop structures represent transcriptional terminators.

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

    Promoter activity in LB broth as measured by chloramphenicol resistance. L. monocytogenes DP-L2986, DP-L2989, and DP-L2812 carrying the hly, actA, and control reverse-orientation actA promoter fusions, respectively, were cultured in buffered LB broth in the absence (light bars) or presence (dark bars) of 10 μg of chloramphenicol per ml. After 5 h, bacterial growth was assessed by measuring the OD600. The data represent the mean ± standard deviation for three individual experiments. The addition of increasing doses of chloramphenicol at the onset of L. monocytogenes10403S culturing in LB broth and measurement of the OD600at various time points demonstrated an MIC of 5 μg/ml (data not shown).

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

    Promoter activity in J774 cells as measured by chloramphenicol resistance. Monolayers of J774 cells grown on glass coverslips were infected with DP-L2986, DP-L2989, or DP-L2812. Chloramphenicol (10 μg/ml) (cm) was added at 2.5 h postinfection. Monolayers were lysed at the indicated time points, and the number of bacteria per coverslip was determined. Results are expressed as the mean number of bacteria ± the standard deviation for three coverslips per time point. One of three experiments with similar results is shown. The MIC of chloramphenicol for L. monocytogenes 10403S grown in J774 cells was determined to be 5 μg/ml by measuring the number of CFU per monolayer in the presence and absence of increasing doses of chloramphenicol at various time points after infection (data not shown).

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

    Promoter activity in LB broth as measured by a β-galactosidase assay. L. monocytogenes DP-L2986 (dark bars), DP-L2989 (hatched bars), and DP-L2812 (see below) carrying thehly, actA, and control reverse-orientationactA promoter fusions, respectively, were cultured in buffered LB broth, and β-galactosidase activity was measured at the indicated time points. Results are expressed as units of β-galactosidase activity per 107 CFU per minute. DP-L2812 background activity, which in no case exceeded 0.08 U, was subtracted from each value. The circles indicate OD600 values for DP-L2986 and DP-L2989, which were identical over 8 h of growth. The data represent the mean ± standard deviation for three individual experiments.

  • Fig. 6.
    • Open in new tab
    • Download powerpoint
    Fig. 6.

    SDS-PAGE of L. monocytogenes secreted and membrane-anchored proteins produced in LB broth. Wild-type L. monocytogenes 10403S was grown in buffered LB broth for 5.5 h, and secreted proteins (lanes 1 and 2) from 10 ml of culture were precipitated with trichloroacetic acid and resuspended in SDS-PAGE sample buffer. Membrane-anchored proteins (lanes 3 to 6) were extracted from bacterial pellets by boiling for 5 min in SDS-PAGE sample buffer. Eighty percent of each sample was subjected to SDS-PAGE, and the gel was stained with Coomassie brilliant blue. Lane 1, 10403S; lane 2, DP-L2161 (10403SΔhly) (14); lane 3, 10403S; lane 4, DP-L1942 (10403SΔactA) (3); lane 5, SLCC-5764 (5); lane 6, DP-L1955 (SLCC-5764ΔactA) (20). Arrows indicate the positions of LLO and ActA. Lanes M show molecular mass standards (kilodaltons [kd]).

  • Fig. 7.
    • Open in new tab
    • Download powerpoint
    Fig. 7.

    Immunoprecipitation of LLO and ActA from L. monocytogenes-infected J774 cells. Bacterial proteins were metabolically labeled during growth in J774 cells or in vitro and immunoprecipitated with monoclonal anti-LLO antibody B3-19 (lanes 1 to 3) or polyclonal anti-ActA antibody 2553 (lanes 4 and 5). Lanes 1 and 4, 10403S-infected J774 cells; lane 2, DP-L2817 (10403S::pfoH438Y)-infected J774 cells; lane 3, supernatant from 10403S cultured in vitro as described in Materials and Methods; lane 5, DP-L1942 (10403SΔactA)-infected J774 cells. An autoradiograph depicting one of two experiments with similar results is shown. The exposure times for the visualization of ActA and LLO by autoradiography were 18 h and 7 days, respectively. For quantitation of ActA and LLO as described in Results, the gel was scanned with a Molecular Dynamics PhosphorImager, and the resulting images were analyzed with ImageQuant software (Molecular Dynamics). Numbers at left are in kilodaltons.

Tables

  • Figures
  • Table 1.

    Promoter activity during bacterial growth in J774 cells

    ExptDP-L2986DP-L2989
    β-Galactosidase activity (U/107CFU/min)a107CFU/dishbβ-Galactosidase activity (U/107 CFU/min)107 CFU/dish
    113.62.431.11.5
    27.12.621.21.6
    315.41.841.81.0
    47.12.632.91.4
    Mean ± SDc10.8 ± 4.331.7 ± 8.4
    • ↵a Background activity, derived from DP-L2812-infected monolayers, was subtracted from each value. β-Galactosidase activity derived from DP-L2812-infected monolayers never exceeded the endogenous β-galactosidase activity produced by uninfected J774 cells.

    • ↵b The number of CFU per dish was determined by lysing infected J774 cells grown on coverslips in duplicate dishes and plating a portion of the lysate on LB agar. The total number of CFU per dish was extrapolated by multiplication by a factor which corrected for the area of the coverslip relative to that of the 60-mm dish.

    • ↵c Derived from four individual experiments. The P value, determined by Student’s t test, for a comparison of the values shown was <0.01.

PreviousNext
Back to top
Download PDF
Citation Tools
Expression of Listeriolysin O and ActA by Intracellular and Extracellular Listeria monocytogenes
Marlena A. Moors, Brian Levitt, Philip Youngman, Daniel A. Portnoy
Infection and Immunity Jan 1999, 67 (1) 131-139; DOI: 10.1128/IAI.67.1.131-139.1999

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.
Expression of Listeriolysin O and ActA by Intracellular and Extracellular Listeria monocytogenes
(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
Expression of Listeriolysin O and ActA by Intracellular and Extracellular Listeria monocytogenes
Marlena A. Moors, Brian Levitt, Philip Youngman, Daniel A. Portnoy
Infection and Immunity Jan 1999, 67 (1) 131-139; DOI: 10.1128/IAI.67.1.131-139.1999
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

Bacterial Proteins
bacterial toxins
Extracellular Space
Heat-Shock Proteins
Hemolysin Proteins
Intracellular Fluid
Listeria monocytogenes
membrane 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