Umeå University's logo

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
Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Show others and affiliations
2019 (English)In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 10, article id 958Article in journal (Refereed) Published
Abstract [en]

The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid’s origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homodimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10’s oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019. Vol. 10, article id 958
Keywords [en]
T4SS, accessory factor, conjugation, relaxosome, X-ray crystallography, protein structural and functional analysis
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-159103DOI: 10.3389/fmicb.2019.00958ISI: 000467035600001PubMedID: 31134011Scopus ID: 2-s2.0-85072734530OAI: oai:DiVA.org:umu-159103DiVA, id: diva2:1316399
Available from: 2019-05-17 Created: 2019-05-17 Last updated: 2024-01-17Bibliographically approved
In thesis
1. Exploring the mechanistic details of Gram-positive Type 4 Secretion Systems
Open this publication in new window or tab >>Exploring the mechanistic details of Gram-positive Type 4 Secretion Systems
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Utforska de mekanistiska detaljerna i Gram-positiva Typ 4 Sekretionssystem
Abstract [en]

Hospital acquired (i.e. nosocomial) infections and antibiotic resistance are large issues in the world today, with about 1.3 million people estimated to have died from antibiotic resistant infections in 2019 alone, and these problems are on the rise. Type 4 Secretion Systems (T4SSs) are complex nanomachineries commonly found on conjugative plasmids. T4SSs are a major route for the translocation of genes encoding for antibiotic resistance and other virulence factors. These systems have primarily been studied in Gram-negative (G-) bacteria even though Gram-positive (G+) bacteria stand for about half of the nosocomial infections. To develop ways to limit the spread of both antibiotic resistance and virulence factors, we need to gain fundamental knowledge of T4SSs in G+ bacteria.

Our work has focused on the conjugative plasmid pCF10 from the G+ bacteria Enterococcus faecalis where all the genes needed for the T4SS are under the regulation of one promoter named PQ. Most G+ T4SSs consist of three groups of proteins, namely the DNA transfer and replication (Dtr) proteins, the channel proteins and the adhesin proteins. In my work, I have focused my attention specifically on i) the regulatory protein PrgU, ii) the Dtr protein PcfF, and iii) the adhesin protein PrgB. These three proteins provide insights into three different parts of the T4SS. PrgU is part of the regulatory process of T4SS expression and has been shown to inhibit cell-toxicity mitigated by PrgB. The Dtr protein PcfF is needed for the formation of the relaxosome complex critical for conjugative transfer of the plasmid, and PrgB is involved in cellular aggregation events and is also a known virulence factor. Interestingly, increased levels of PrgB have been shown to be toxic to the cells. To inhibit PrgB induced cell toxicity, its production needs to be tightly regulated.

The aims of my PhD thesis were to examine conjugation complexes belonging to Type 4 Secretion Systems in Gram-positive bacteria and to determine their function, molecular structures, and regulation. By using a combination of in vivo and in vitro methods we have; i) showed that PrgU binds to the IGR located downstream of the PQ promoter, and that the deletion of prgU in pCF10 containing cells produces increased mRNA levels of the full prgQ transcript, ii) solved the crystal structure of PcfF and identified residues that are important for the interaction with the relaxase and the origin of transfer (oriT) DNA in vitro, and confirmed this by biochemical assays and, iii) solved the entire structure of PrgB using a combination of X-ray crystallography and cryo-EM and performed in vivo assays to confirm its functions.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2022. p. 61
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2186
Keywords
Antimicrobial resistance, Horizontal gene transfer, Gram-positive bacteria, Type 4 Secretion Systems, Conjugation, Enterococcus faecalis, pCF10, PrgU, PcfF, PrgB
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-194545 (URN)978-91-7855-815-5 (ISBN)978-91-7855-814-8 (ISBN)
Public defence
2022-06-03, Hörsal UB.A.240 – Lindellhallen 4, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2022-05-13 Created: 2022-05-10 Last updated: 2023-06-03Bibliographically approved

Open Access in DiVA

fulltext(8732 kB)207 downloads
File information
File name FULLTEXT01.pdfFile size 8732 kBChecksum SHA-512
b32e53381e1e23751d1ae929f7f4d9235bce56080680e400c23040fc06d52d6e6d62dc2687d83c4d6766dc9213fb3322844ed39037e8cfa13b2806127f018da7
Type fulltextMimetype application/pdf

Other links

Publisher's full textPubMedScopus

Authority records

Rehman, SaimaSchmitt, AndreasBerntsson, Ronnie P. -A.

Search in DiVA

By author/editor
Rehman, SaimaSchmitt, AndreasLassinantti, LenaBerntsson, Ronnie P. -A.
By organisation
Department of Medical Biochemistry and Biophysics
In the same journal
Frontiers in Microbiology
Structural Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 207 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 570 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