umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Interaction between the PapI protein and the alpha subunit of Escherichia coli RNA polymerase
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Bernt Eric Uhlin)
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Bernt Eric Uhlin)
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Uropathogenic Escherichia coli (UPEC) expressing P fimbriae (also denoted Pap or P pili) have the ability to adhere in a specific manner to uroepithelial cells in the cause of urinary tract infections. P fimbriae expression is subject to an intricate regulatory mechanism playing an important role in pathogenesis, allowing bacteria to sense its environment and express fimbriae when needed, and turn off the expression when it may be unnecessary or detrimental to the bacteria. The expression of P fimbriae in the uropathogenic E. coli depends on transcription of the major pap operon in a gene cluster that consists of the polycistronic papB operon and the divergently orientated monocistronic papI operon. The transcription of pap is positively controlled by the local transcription factors PapI (8 kDa) and PapB (12 kDa) in concert with the global regulators leucine-responsive regulatory protein (Lrp) and the cAMP receptor protein (CRP). DNA adenine methylase (Dam) activity is also required for pap transcription. PapI is known to interact with Lrp and its role in relation to the transcriptional activation of pap is thought to be indirect without contact with the RNA polymerase. The CRP-cAMP complex is known to activate the expression of pap via direct contact with the alpha (α) subunit of E. coli RNA polymerase. Methodology/Principal findings: Here, we show that an excess of α protein inhibits the transcription of pap as revealed by using a collection of strains which carry plasmids expressing α subunits with different point mutations in the α subunit of E. coli RNA polymerase. The inhibition was partially restored by the introduction of a papI clone expressing higher level of PapI. Experiments with purified proteins verified that PapI physically interacts with the α subunit of E. coli RNA polymerase in vitro. Conclusion/Significance: Our findings indicate that the PapI protein may interact directly with the RNA polymerase complex and that such interactions need to be considered in its role as a transcription factor in the activation of pap transcription.

Keyword [en]
Escherichia coli, PapI, alpha subunit, RNAP
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-27738OAI: oai:DiVA.org:umu-27738DiVA: diva2:277441
Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2009-11-26Bibliographically approved
In thesis
1. Molecular analysis of transcription factors in uropathogenic E. coli adhesin operons
Open this publication in new window or tab >>Molecular analysis of transcription factors in uropathogenic E. coli adhesin operons
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Molekylär analys av transkriptionsfaktorer i adhesin operon hos uropatogena E. coli
Abstract [en]

The main causative agent of human urinary tract infections is the uropathogenic Escherichia coli (UPEC) pathotype. It may cause disease due to its ability to express a number of bacterial virulence factors. Fimbrial adhesins are particularly important for the initial establishment of infection in the urinary tract. The fimbriae are hair-like structures protruding from the bacterial cell and by attaching to specific receptors in the urinary tract they mediate adherence to different cell types, allowing the bacteria to resist the shear forces from urine flow. The UPEC strains generally carry multiple determinants for fimbrial adhesins. Previous studies have indicated that there is a co-regulation between different fimbrial genes and one factor that has been implicated in this is the PapB protein, acting as a transcriptional regulator of P-fimbrial expression. The PapB protein can be regarded as the prototype of a family of fimbrial regulators that show high homology between different fimbrial operons. One homolog is FocB, regulator of F1C fimbriae. In this study, the role of the FocB protein in the regulation of F1C fimbriae as well as in the co-regulation with other fimbrial genes was investigated. It was observed that FocB binds to DNA, similarly to PapB, in an oligomeric fashion and that PapB and FocB can form hetero-oligomeric complexes, which appear to have a repressive role in the regulation of the F1C fimbriae. In addition, the FocB protein also had a repressive effect on transcription of the fim operon, which encodes theType 1 fimbriae. For further analysis of FocB in vitro, we developed efficient procedures for purification of the protein and established conditions for its crystal formation with the aim to conduct X-ray diffraction studies. By the hanging-drop vapour-diffusion method, we obtained crystals that in the X-ray analysis diffracted sufficiently well to allow modelling of a high resolution structure of FocB. The structural model was considered in relation to the DNA binding properties of the protein. The FocB analysis represents the first structural model of this family of transcriptional factors. This model should aid in further understanding of the roles and functions of these proteins in the regulation of the UPEC fimbrial operons. The complexity of the system, with multiple factors involved in the regulation of fimbrial operons, was revealed in earlier studies of the PapI protein showing that PapI activates transcription of the pap operon as a part of a complex with the global regulator Lrp. However, PapI itself did not appear to bind to DNA and its mode of action has remained unclear. By genetic analyses and in vitro studies we show that PapI may interact also with the α subunit of the RNA polymerase. This finding indicates that PapI might directly interact with the transcriptional apparatus and thus aid in the activation of pap expression. Bacteria are frequently releasing outer membrane vesicles (OMVs) from their surface. We studied the release of the haemolysin toxin from E. coli in connection with formation of OMVs and found that the toxin was tightly associated with the vesicles in an active form. By overproduction of the PapB or PapI regulators in order to maximise the population of bacteria expressing fimbriae, we could detect P fimbriae proteins associated with OMVs that displayed specific adhesion to receptor-coated beads. This suggests a possible scenario in which the vesicles canfunction as directed vehicles of bacterial virulence factors.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2009. 63 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1304
Keyword
UPEC, E. coli, F1C, P fimbriae, cross-talk, FocB, PapB, crystal structure, OMVs
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-27757 (URN)978-91-7264-879-1 (ISBN)
Public defence
2009-12-16, Major Groove, Institutionen för Molekylärbiologi, Byggn. 6L, Norrlands Universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2009-11-26 Created: 2009-11-19 Last updated: 2011-04-28Bibliographically approved

Open Access in DiVA

No full text

By organisation
Molecular Biology (Faculty of Medicine)
Microbiology in the medical area

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 80 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf