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YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 27, 19089-19097 p.Article in journal (Refereed) Published
Abstract [en]

Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.

Place, publisher, year, edition, pages
2014. Vol. 289, no 27, 19089-19097 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-106016DOI: 10.1074/jbc.M113.541672ISI: 000339062900039OAI: oai:DiVA.org:su-106016DiVA: diva2:734601
Available from: 2014-07-18 Created: 2014-07-18 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Protein trafficking in the cell envelope of Escherichia coli: Identification and characterisation of a novel chaperone
Open this publication in new window or tab >>Protein trafficking in the cell envelope of Escherichia coli: Identification and characterisation of a novel chaperone
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell envelope of Gram-negative bacteria, like Escherichia coli, is composed of a cytoplasmic membrane, a periplasmic space containing a peptidoglycan layer and an outer membrane. About 30 % of all proteins are localised in the cell envelope. These proteins have to be inserted into or translocated across the inner membrane by the SecYEG translocon. They are then chaperoned to their final destination by a network of chaperones. The broad aim of this work was to provide a better understanding of protein trafficking through the bacterial cell envelope. We have identified a novel membrane protein complex consisting of the periplasmic chaperone PpiD and the uncharacterised protein YfgM. Both are anchored in the inner membrane and have periplasmic domains. By co-immunoprecipitations and two-dimensional gel electrophoresis it could be demonstrated that YfgM and PpiD form a supercomplex with the SecYEG translocon. Furthermore, a chemical-genetic approach showed that YfgM is part of the periplasmic chaperone network that is essential for envelope protein biogenesis. Moreover, it could be shown that YfgM is required for the stability of the periplasmic chaperone HdeB. Finally, evidence that YfgM might also be involved in the lateral insertion of transmembrane domains was provided. In summary, this thesis details the identification and characterisation of a novel ancillary subunit of the SecYEG translocon that is involved in the periplasmic chaperone network in the cell envelope of Escherichia coli.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 73 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-106277 (URN)978-91-7447-944-7 (ISBN)
Public defence
2014-09-25, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2014-09-03 Created: 2014-07-30 Last updated: 2017-10-30Bibliographically approved
2. Antibiotic uptake in Gram-negative bacteria
Open this publication in new window or tab >>Antibiotic uptake in Gram-negative bacteria
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The increasing emergence and spread of antibiotic-resistant bacteria is a serious threat to public health. Of particular concern are Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant to a large number of antibiotics and thus our treatment options are rapidly declining. In addition to the increasing number of antibiotic-resistant bacteria, a major problem is that many of the antibiotics at our disposal are ineffective against Gram-negative bacteria. This is partly due to the properties of the outer membrane (OM) which prevents efficient uptake. The overarching goal of this thesis was to investigate how the OM of the Gram-negative bacterium E. coli could be weakened to improve the activity of antibiotics.

In the first two papers of my thesis (paper I + II), I investigated the periplasmic chaperone network which consists of the two parallel pathways SurA and Skp/DegP. This network is essential for the integrity of the OM and strains lacking either SurA or Skp are defective in the assembly of the OM, which results in an increased sensitivity towards vancomycin and other antimicrobials. We identified a novel component of the periplasmic chaperone network, namely YfgM, and showed that it operates in the same network as Skp and SurA/DegP. In particular, we demonstrated that deletion of YfgM in strains with either a ΔsurA or Δskp background further compromised the integrity of the OM, as evidenced by an increased sensitivity towards vancomycin.

In the remaining two papers of my thesis (paper III + IV), the goal was to characterize small molecules that permeabilize the OM and thus could be used to improve the activity of antibiotics. Towards this goal, we performed a high-throughput screen and identified an inhibitor of the periplasmic chaperone LolA, namely MAC-13243, and showed that it can be used to permeabilize the OM of E. coli (paper III). We further demonstrated that MAC-13243 can be used to potentiate the activity of antibiotics which are normally ineffective against E. coli. In the last paper of my thesis (paper IV), we undertook a more specific approach and wanted to identify an inhibitor against the glycosyltransferase WaaG. This enzyme is involved in the synthesis of LPS and genetic inactivation of WaaG results in a defect in the OM, which leads to an increased sensitivity to various antibiotics. In this paper, we identified a small molecular fragment (compound L1) and showed that it can be used to inhibit the activity of WaaG in vitro.

To summarize, this thesis provides novel insights into how the OM of the Gram-negative bacterium E. coli can be weakened by using small molecules. We believe that the two identified small molecules represent important first steps towards the design of more potent inhibitors that could be used in clinics to enhance the activity of antibiotics.

Place, publisher, year, edition, pages
Department of Biochemistry and Biophysics, Stockholm University, 2017
Keyword
Gram-negative bacteria, Antibiotic uptake, Outer membrane, Lipopolysaccharide
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-148541 (URN)978-91-7797-041-5 (ISBN)978-91-7797-042-2 (ISBN)
Public defence
2017-12-12, Magnélisalen Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2017-11-17 Created: 2017-10-29 Last updated: 2017-11-17Bibliographically approved

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