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 Pathogenesis

Characterization of sarR, a Modulator ofsar Expression in Staphylococcus aureus

Adhar Manna, Ambrose L. Cheung
Adhar Manna
Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ambrose L. Cheung
Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/IAI.69.2.885-896.2001
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

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

    (A) Schematic of the sar promoters and transcripts analyzed in this study. Positions of the transcription start sites (146, 409, and 711 bp upstream of the translation start) for P1, P3, and P2 promoters are depicted according to published sequence (2). The P1, P3, and P2 transcripts have previously been designated a sarA, sarC, andsarB transcripts. The 49-bp sequence outlined was used to construct a DNA-specific column as described elsewhere (27). Relative positions of the sar promoter fragments used in gel shift and footprinting studies are indicated (filled boxes); the promoter fragments for the GFP transcriptional fusion assays are represented by empty boxes. (B) Promoter region ofsarR. The transcription start site has been mapped by primer extension (data not shown) to position 119. The putative −10 and −35 promoter boxes are in bold and underlined. (C) Alignment of SarR with SarA. Colons represent identity; periods indicate conservative substitutions.

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

    Purification of SarR from the pET11b expression vector. Equivalent volumes of protein fractions obtained during the purification process were applied to an SDS–12% polyacrylamide gel. Lane 1, whole-cell lysate of E. coli containing pALC1357 (pET11b with the sarR gene); lane 2, supernatant of the cell lysate after clarification by centrifugation; lane 3, supernatant before 40% ammonium sulfate precipitation; lane 4, pellet resulting from 40% ammonium sulfate precipitation; lane 5, pellet from 80% ammonium sulfate precipitation; lane 6, flowthrough of the redissolved 80% ammonium sulfate precipitant as applied to a MonoQ column (Pharmacia); lane 7, flowthrough from the MonoS column (Pharmacia); lane 8, NaCl elution from the MonoS column. N-terminal sequencing confirmed the identity of the purified SarR protein.

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

    PCR amplification of sarR-like genes in S. aureus strains RN6390, Cowan I, DB, and Newman, S. epidermidis, S. haemolyticus, and S. saprophyticus using primers 5′-208ATGAGTAAAATTAATGATATTAAT231-3′ and 5′-589TCGTTCAATGTTATTAAACG569-3′. (B) Southern blot of the above strains, restricted withClaI and probed with a 345-bp sarR probe (nt 208 to 552). (C) Northern blot of total cellular RNA (10 μg each) of the above strains, probed with a sarR probe. (D) Cell lysates of the above strains, immunoblotted onto nitrocellulose and probed with anti-SarR monoclonal antibody 2A7 at a 1:2,000 dilution.

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

    Gel shift assay of end-labeled 32P fragment of sar P1 (nt 531 to 859) 2, P2 (nt 1 to 196), and P3 (nt 364 to 525) promoters. Increasing amounts (30, 60, 100, 150, 200, 250, and 300 ng) of purified SarR were applied to the reaction mixtures. In competition assays, 50- and 100-fold excesses of unlabeled DNA fragments were added.

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

    DNase I footprinting assays of SarR with end-labeled32P sar P2 (49-bp fragment), P1 (nt 531 to 859), and P1′ (nt 620 to 859) promoter fragments. The sequence was deduced from G+A ladder reactions run in parallel by the standard method (26). The following amounts of SarR were applied to thesar P2 and P1 reactions: 30, 60, and 100 ng. Withsar P1′, only lanes containing 30 and 60 ng of SarR protein are shown. The binding site of SarR on the sar P3 promoter (underlined) was also mapped: 373TTACTAAATTAAAAAAATTA402(footprinting data not shown) (2). The underlined nucleotides represent the 7- or 8-base sequence conserved in the binding site throughout the sar promoter region.

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

    Expression of sarR in parental strain RN6390 and its sar (ALC488) and agr (RN6911) mutants. (A) Northern blots of sarR transcript in RN6390 and its isogenic sar and agr mutants. Ten micrograms of total cellular RNA was applied to each lane. The sarR probe was a 345-bp fragment (nt 208 to 552). OD650 of 0.7, 1.1, and 1.7 represent mid-log, late log, and early stationary phases, respectively, as predicted from the growth cycle. (B) Ethidium bromide stain of the above RNA gel prior to transfer to a hybridization membrane. (C) Expression of SarR on an immunoblot probed with anti-SarR antibody 2C7. Each lane contains 25 μg of cell extract of RN6390 grown to late log and early stationary phases. Cells at mid-log phase expressed little SarR; as expected, SarR was not detected in thesarR mutant ALC1713 (data not shown). The positive control lane contains 0.1 μg of purified SarR protein.

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

    Promoter activation of sar P1 and combined P2-P3-P1 promoters fused to a gfpuvrreporter gene as evaluated in a fluorescence spectrophotometer (FL600; BioTek Instruments). (A) Recombinant shuttle plasmid pALC1484 containing thesar P1 promoter linked to gfpuvr(excitation maxima at 488 nm) was introduced into strain RN6390 (♦) and its isogenic sarR mutant ALC1713 (•). A negative control (RN6390 containing pALC1484 with no promoter fragment) showed no significant background fluorescence (background = ∼300 fluorescence units [data not shown]). Cells were obtained hourly (200 μl of each in duplicate) during the growth cycle (from h 2 to 10 after an initial dilution of 1:100 in fresh medium) to obtain fluorescence and OD values in the same instrument. To minimize variations in fluorescence attributable to cell density, the data are presented as average of reported fluorescence per OD unit in triplicate samples plotted against the mean OD. The error bar was too small to be discerned (typically less than 100 fluorescence units). The experiment was repeated at least thrice; one representative experiment is shown. (B) Plot similar to that in panel A except that the combinedsar P2-P3-P1 promoter fragment was used in place of the P1 promoter in the recombinant pALC1484 containing thegfpuvrreporter gene. In similar assays with the individual sar P2 and P3 promoters linked to thegfpuvrreporter in the isogenic pair, we detected no differences in GFPuvr expression between the parental strain and the sarR mutant. However, the level of fluorescence associated with individual P2 and P3 promoters was very low and only slightly above background levels.

  • Fig. 8.
    • Open in new tab
    • Download powerpoint
    Fig. 8.

    Effect of the sarR mutation on SarA andagr expression. (A) SarA expression during mid-log, late log, and early stationary phases; (B) agrA (RNAII) transcription. (A) Immunoblot of cell extracts (5 μg of protein each) of RN6390 and the sarR mutant (harvested at mid-log, late log, and stationary phases) probed with anti-SarA monoclonal antibody 1D1 at 1:2,000 dilution. The positive control lane contains 0.5 μg of purified SarA. Similar results were obtained with 25 μg of protein per lane. (B) Northern blot of the RNAII (agrA probe) transcript in RN6390 and the sarR mutant (10 μg of total RNA each). The agrA probe corresponds to nt 3830 to 4342 according to the published sequence 23.

Tables

  • Figures
  • Table 1.

    Strains and plasmids used

    Strain or plasmid Reference or source Comments
    S. aureus
     RN422032Mutant of 8325–4 that accepts foreign DNA
     RN6390 32 Laboratory strain that maintains its hemolytic pattern when propagated on sheep erythrocyte agar (parental strain)
     RN6911 34 agr mutant of RN6390 with Δagr::tetM mutation
     ALC4889 sar mutant with sarA::ermCmutation
     ALC1713 This study sarR mutant of RN6390 with ΔsarR::ermC mutation
     Cowan I17 Laboratory strain
     DB 10 Clinical blood isolate previously used in adhesion and endocarditis studies
     Newman29 Laboratory strain
    S. epidermidisStrain from collection at Utrecht University Hospital
    S. haemolyticusStrain from collection at Utrecht University Hospital
    S. saprophyticusStrain from collection at Utrecht University Hospital
    E. coli
     XL-1 Blue 26 Host strain for cloning
     DH5α 26 Host strain for cloning
    Plasmids
     pCR2.1 Invitrogen E. coli cloning vector for direct cloning of PCR products
     pBluescript Stratagene E. coli cloning vector
     pUC18 26 E. coli cloning vector
     pACYC177New England Biolabs E. coli cloning vector
     pCL52.1 24 Temperature-sensitive E. coli-S. aureus shuttle vector
     pET11b NovagenExpression vector for E. coli
     pALC926 This study pUC18 containing a 49-bp fragment upstream of the P2 promoter of the sar locus
     pALC1357 This study pET11b containing the 345-bp sarR gene at theNdeI/BamHI site
     pALC1361 This studypACYC177 with a ∼4-kb ClaI fragment containing thesarR region of RN6390
     pALC1627 This studypBluescript with a 2.5-kb EcoRI/ClaI fragment containing the sarR gene subcloned from pALC1361
     pALC1687 This study pBluescript with a 290-bp deletion of the sarR gene in pALC1627
     pALC1696 This studypCL52.1 with a 290-bp sarR deletion replaced by theermC gene at the EcoRV/SalI site
PreviousNext
Back to top
Download PDF
Citation Tools
Characterization of sarR, a Modulator ofsar Expression in Staphylococcus aureus
Adhar Manna, Ambrose L. Cheung
Infection and Immunity Feb 2001, 69 (2) 885-896; DOI: 10.1128/IAI.69.2.885-896.2001

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.
Characterization of sarR, a Modulator ofsar Expression in Staphylococcus aureus
(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
Characterization of sarR, a Modulator ofsar Expression in Staphylococcus aureus
Adhar Manna, Ambrose L. Cheung
Infection and Immunity Feb 2001, 69 (2) 885-896; DOI: 10.1128/IAI.69.2.885-896.2001
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
Gene Expression Regulation, Bacterial
Genes, Regulator
Staphylococcus aureus
Trans-Activators

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