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 Cart

Main menu

  • Home
  • Articles
    • Archive
  • About the Journal
    • About EC
    • For Librarians
    • For Advertisers
    • FAQ
  • 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 Cart

Search

  • Advanced search
Eukaryotic Cell
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Archive
  • About the Journal
    • About EC
    • For Librarians
    • For Advertisers
    • FAQ
Articles

Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and d-Xylose Metabolism in Hypocrea jecorina

Astrid R. Stricker, Karin Grosstessner-Hain, Elisabeth Würleitner, Robert L. Mach
Astrid R. Stricker
Gene Technology, Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9/166/5/2, A-1060 Wien, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Karin Grosstessner-Hain
Gene Technology, Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9/166/5/2, A-1060 Wien, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Elisabeth Würleitner
Gene Technology, Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9/166/5/2, A-1060 Wien, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert L. Mach
Gene Technology, Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Getreidemarkt 9/166/5/2, A-1060 Wien, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: rmach@mail.zserv.tuwien.ac.at
DOI: 10.1128/EC.00211-06
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Additional Files
  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    (A) Growth of parental strain QM9414, the xyr1 retransformation strain (Rexyr1A), and the Δxyr1 strain on plates containing MA medium supplemented with 1% (wt/vol) l-arabinose (AN), l-arabitol (AL), xylitol (XL), and d-xylose (XO) and 2% (wt/vol) agar-agar at 30°C after 4 days. (B) Schematic presentation of the d-xylose catabolic pathway. In H. jecorina, d-xylose cannot be directly converted into d-xylulose because fungi do not have xylulose isomerase at their disposal (51).

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

    Enzyme activities of d-xylose reductase (A and C) and l-arabinose reductase (B and D) given in U/g, measured in cell extracts of QM9414 (QM) and the Δxyr1 strain. All strains were precultured in MA medium containing 1% (wt/vol) glycerol. For analysis of both reductase activities, mycelia were transferred to MA medium containing 1% (wt/vol) d-xylose (XO) (A and D) and l-arabinose (AN) (B and C) and grown for 5 and 10 h. One unit of activity is defined as the amount of enzyme responsible for the consumption of 1 micromole NADPH per minute due to its oxidation in the presence of d-xylose or l-arabinose under the defined assay conditions (5). Values are the means of results from three independent experiments. Error bars indicate standard deviations.

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

    Relative transcription levels of cbh1 (A), cbh2 (B), and egl1 (C). The parental strain QM9414 (QM) and the Δxyr1 strain were precultured with glycerol and thereafter transferred to MA medium containing 2 mM sophorose (SO) as inducer or 1% (wt/vol) cellulose (CL) as the sole carbon source and incubated 5 and 8 h or 8 and 24 h, respectively. After parallel extraction of RNA and DNA and reverse transcription of RNA, real-time PCR analysis of the respective genes was performed. Values are the means of results from three independent experiments. Error bars indicate standard deviations.

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

    Xylanase activities in culture supernatants of the parental strain QM9414 and the Δxyr1 strain. Both strains were precultured in MA medium containing 1% (wt/vol) glycerol. Detection of xylanase activity formation was accomplished after transfer of equal amounts of wet weight of mycelia to MA medium without a carbon source or to medium containing 2 mM sophorose or xylobiose as inducer and incubated for 5 and 8 h, or to medium containing 1% (wt/vol) d-xylose or glucose grown for 5 and 8 h, or to MA medium containing 1% (wt/vol) oat spelt xylan or carboxymethylcellulose and grown for 8 and 24 h. One unit of activity is defined as the amount of enzyme required to release 1 micromole of xylose reducing sugar equivalents per minute at 40°C. Data are the means of results from three independent experiments. Error bars indicate standard deviations.

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

    (A) Total (extracellular and mycelial-bound) β-glucosidase activity (+) and activity in the culture supernatant (−) of the parental and the Δxyr1 strains after replacement of glycerol pregrown mycelia into medium containing 2 mM sophorose (SO) and incubation for 5 and 8 h. (B) Activity of β-xylosidase in supernatants of the parental and the Δxyr1 strains after replacement of glycerol pregrown mycelia into medium containing 2 mM xylobiose (XB) and incubation for 5 h. All data are the means of results from three independent experiments. Error bars indicate standard deviations.

Tables

  • Figures
  • Additional Files
  • TABLE 1.

    Primers and probes used throughout the study

    Primer nameSequence (5′-3′)Employed for
    ActfTGAGAGCGGTGGTATCCACGActin real-time PCR
    ActrGGTACCACCAGACATGACAATGTTGActin real-time PCR
    bgl1fCAGACAGTCACTCAACATCGGG bgl1 real-time PCR
    bgl1rACCTTATCTTGGAGATTGAGCTTTGCC bgl1 real-time PCR
    bgl2fACGGCTGCCTACCAGATCG bgl2 real-time PCR
    bgl2rAGCCGTCGGCGATCTTGC bgl2 real-time PCR
    bxl1fGCCAACTTCGCCACCAAGG bxl1 real-time PCR
    bxl1rCGGCAATCTGGTGGATCAATGTG bxl1 real-time PCR
    TaqMan cbh1 FAMCTGGACTCCACCTACCCGACAAACGAGACC cbh1 real-time PCR
    chb1fGATGATGACTACGCCAACATGCTG cbh1 real-time PCR
    cbh 1Df2TGTTACAGTACTACGCCAACATGCTG cbh1 real-time PCR
    cbh 1rACGGCACCGGGTGTGG cbh1 real-time PCR
    TaqMan cbh2 TRGACTTGCCGGATCGCGATTGCGCTGCCC cbh2 real-time PCR
    cbh2fCTATGCCGGACAGTTTGTGGTG cbh2 real-time PCR
    cbh2DrGTCAGGCTCTGGAAGAAGG cbh2 real-time PCR
    cbh2rGTCAGGCTCAATAACCAGGAGG cbh2 real-time PCR
    TaqMan egl1 FAMCAACTCGAGGGCGAATGCCTTGACCCCTCACTC egl1 real-time PCR
    egl1fCTGCAACGAGATGGATATCCTGG egl1 real-time PCR
    egl1DrGAGAGCGCCAGGAAAGGG egl1 real-time PCR
    egl1rGTAGTAGCTTTTGTAGCCGCTGC egl1 real-time PCR
    XorfCTGTGACTATGGCAACGAAAAGGAG xyl1 real-time PCR
    XorrCACAGCTTGGACACGATGAAGAG xyl1 real-time PCR
    TaqMan xyn1 FAMCGTCCAACCAACGCCCACAACAA xyn1 real-time PCR
    Taqxyn1fCAGCTATTCGCCTTCCAACAC xyn1 real-time PCR
    Taqxyn1DrGAGGAGTCCTCCTACGCAGAA xyn1 real-time PCR
    Taqxyn1rCCAAAGTTGATGGGAGCAGAA xyn1 real-time PCR
    TaqMan xyn2 FAMCTGCCATCCCTTGCCGCC xyn2 real-time PCR
    Taqxyn2fGGTCCAACTCGGGCAACTTT xyn2 real-time PCR
    Taqxyn2DrGGTAAGGGTAGGTAGTCTTACTTGTTC xyn2 real-time PCR
    Taqxyn2rCCGAGAAGTTGATGACCTTGTTC xyn2 real-time PCR
    TaqMan xyr1 FAMCGCGCTTGTGACCAGTGCAACCAGCTTCGTACC xyr1 real-time PCR
    xyr1fCCCATTCGGCGGAGGATCAG xyr1 real-time PCR
    xyr1DrGATCAGTACATGTGCTTGAGCGC xyr1 real-time PCR
    xyr1rCGAATTCTATACAATGGGCACATGGG xyr1 real-time PCR
    Xyr1.1.fGGTACCAATTGTGAGCGCATCACConstruction of deletion cassette
    Xyr1.2.rCTGTCGACGATGGGAATTCGGGTCAAATGACConstruction of deletion cassette
    Xyr2.1.fCCGAATTCCCATCGTCGACAGGCACCTGGConstruction of deletion cassette
    Xyr2.2.rGGTACCGAACATAGCCCAACGConstruction of deletion cassette
  • TABLE 2.

    Growth of the parental strain and strains Δxyr1 and Rexyr1 on plates containing MA medium supplemented with different carbon sourcesa

    Avg mycelial growth zone diam (cm)b
    Carbon sourceAfter 3 daysAfter 6 days
    QM9414Δxyr1Rexyr1AQM9414Δxyr1Rexyr1A
    Glucose3.7 ± 0.293.9 ± 0.173.5 ± 0.198.0 ± 0.008.0 ± 0.008.1 ± 0.04
    Glycerol3.0 ± 0.153.4 ± 0.003.2 ± 0.127.2 ± 0.357.1 ± 0.107.2 ± 0.12
    l-(+)-Arabinose3.3 ± 0.103.5 ± 0.213.5 ± 0.177.4 ± 0.127.5 ± 0.407.3 ± 0.15
    l-(−)-Arabitol3.1 ± 0.173.7 ± 0.063.3 ± 0.087.7 ± 0.157.7 ± 0.127.6 ± 0.22
    d-(+)-Xylose4.1 ± 0.231.1 ± 0.104.2 ± 0.218.0 ± 0.002.4 ± 0.068.2 ± 0.16
    Xylitol3.3 ± 0.123.5 ± 0.063.5 ± 0.127.5 ± 0.067.5 ± 0.007.6 ± 0.17
    Xylan2.0 ± 0.172.5 ± 0.122.2 ± 0.195.5 ± 0.384.6 ± 0.125.6 ± 0.16
    Cellulose2.8 ± 0.173.3 ± 0.173.0 ± 0.145.7 ± 0.106.0 ± 0.005.9 ± 0.11
    • ↵ a MA medium (1% wt/vol) cultured at 30°C.

    • ↵ b Values are the means ± SD of results from three independent experiments.

  • TABLE 3.

    Relative xyn1 transcription levels of the wild-type and the Δxyr1 strains

    Carbon source (cultivation time, h)Amt of mRNA (cDNA) per gene dosea
    QM9414Δxyr1
    No carbon source (3)1.0 ± 0.5NDb
    No carbon source (5)0.5 ± 0.1ND
    No carbon source (8)0.4 ± 0.2ND
    Glucose (3)NDND
    Glucose (5)NDND
    Xylose (3)1.9 ± 0.4ND
    Xylose (5)169.3 ± 16.5ND
    Xylose (8)0.0 ± 0.0ND
    Xylan (8)16.1 ± 2.6ND
    Xylan (24)9.8 ± 0.0ND
    No carbon source (3)1.0 ± 0.1ND
    No carbon source (5)3.8 ± 0.5ND
    No carbon source (8)NDND
    Sophorose (5)97.9 ± 9.6ND
    Sophorose (8)6,701.7 ± 531.1ND
    Xylobiose (3)6,085.9 ± 637.1ND
    Xylobiose (5)265.6 ± 11.2ND
    Xylobiose (8)37.5 ± 3.2ND
    Xylan (8)4,305.9 ± 415.3ND
    Xylan (24)3,819.4 ± 277.6ND
    • ↵ a Values are the means ± SD of results from three independent experiments.

    • ↵ b ND, no detection.

  • TABLE 4.

    Xylanase and cellulase activities in supernatants from direct cultivations of the parental strain and the Δxyr1 strains with xylan or cellulose as respective carbon sources

    Cultivation time (h)Xylanase activity (U/μg protein) in xylan supernatantsa,cCellulase activity (U/μg protein) in cellulose supernatantsb,c
    QM9414Δxyr1QM9414Δxyr1
    24NDdND0.003 ± 0.000ND
    480.206 ± 0.015ND0.655 ± 0.012ND
    720.142 ± 0.009ND0.885 ± 0.022ND
    • ↵ a One unit of activity is defined as the amount of enzyme required to release 1 micromole of xylose reducing sugar equivalents per min under the defined assay conditions.

    • ↵ b One unit of activity is defined as the amount of enzyme required to release 1 micromole of glucose reducing sugar equivalents per min under the defined assay conditions.

    • ↵ c Values are the means ± SD of results from three independent experiments.

    • ↵ d ND, no detection.

  • TABLE 5.

    Relative transcript levels of bgl1, bgl2, and bxl1 in the wild-type and the Δxyr1 strains

    Analyzed gene, carbon source, and incubation time (h)Transcript ratioa,b
    QM9414Δxyr1
    bgl1, sophorose (8)1.0 ± 0.1NDc
    bgl2, sophorose (8)1.3 ± 0.21.1 ± 0.0
    bxl1, xylobiose (5)58.2 ± 0.4ND
    • ↵ a Values were calculated with reference to analyses of QM9414, bgl1, sophorose cultivated for 8 h.

    • ↵ b Values are means ± SD from results of three independent experiments.

    • ↵ c ND, no detection.

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Regulatory impact of Xyr1 on hydrolase expression in T. reesei.
      Zipped TIF document, 206K.
PreviousNext
Back to top
Download PDF
Citation Tools
Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and d-Xylose Metabolism in Hypocrea jecorina
Astrid R. Stricker, Karin Grosstessner-Hain, Elisabeth Würleitner, Robert L. Mach
Eukaryotic Cell Dec 2006, 5 (12) 2128-2137; DOI: 10.1128/EC.00211-06

Citation Manager Formats

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

Email

Thank you for sharing this Eukaryotic Cell 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.
Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and d-Xylose Metabolism in Hypocrea jecorina
(Your Name) has forwarded a page to you from Eukaryotic Cell
(Your Name) thought you would be interested in this article in Eukaryotic Cell.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and d-Xylose Metabolism in Hypocrea jecorina
Astrid R. Stricker, Karin Grosstessner-Hain, Elisabeth Würleitner, Robert L. Mach
Eukaryotic Cell Dec 2006, 5 (12) 2128-2137; DOI: 10.1128/EC.00211-06
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
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Endo-1,4-beta Xylanases
Hypocrea
Xylose

Related Articles

Cited By...

About

  • About EC
  • For the Media
  • For Librarians
  • For Advertisers
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • Submit a Manuscript to mSphere

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: 1535-9778; Online ISSN: 1535-9786