Trans-Membrane Trafficking Unit

Associate Professor Fadel Samatey

Abstract

Motility is a very important function in the living world. For this purpose, organisms such as bacteria have developed one of the most incredible molecular machines: the flagellar system. Bacteria such as Escherichia coli and Salmonella typhimurium swim by rotating long helical filaments called the flagellum. The flagellum is a complex structure made by the association of many different proteins. It can be divided into three parts: 1) the filament: a long tubular structure that works as a helical propeller, 2) the hook: a short, highly flexible tubular segment that works as a universal joint, 3) the basal body: a rotary motor embedded in the cell membrane and the protein export apparatus attached. The basal body is made of about 20 different proteins while the hook and the filament are made of single proteins, the hook protein (FlgE) and flagellin (FliC), respectively. The hook and the filament are joined together by two junction proteins, the hook associated protein 1 (HAP1) and 3 (HAP3). The filament is capped by the hook associated protein 2 (HAP2), which is essential for the filament polymerization. The flagellar motor can be divided into two major components: the stator and the rotor. The stator is made of multiple copies of two integral membrane proteins, MotA and MotB, and works as a proton channel. The rotor consists of multiple copies of proteins FliF and FliG, and the C ring is attached on its cytoplasmic side to regulate the rotational direction. It is thought that the torque is generated by the interactions between FliG and the MotA/B complex To understand the mechanisms of how this nano-machine works, the complete structural information of the flagellum at atomic level is necessary. All the bacterial flagellar axial proteins are exported to its growing end through a 2 -3 nm channel located in its centre. The protein export system attached at the cytoplasmic side of the basal body is called the type III export apparatus. To date there have been little high-resolution work done on the T3SS. In the case of the bacterial flagellum, the export system is made by six membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, FliR, and three cytoplasmic proteins: FliI, FliH and FliJ.

The T3SS of the bacterial flagellum is homologous to the one found in Gram-negative pathogenic bacteria, which secretes virulence factors to host cells for invasion that triggers diseases. The type III secretion systems are present in both the animal and plant pathogenic bacteria and they share a number of core structural components that are highly conserved. The needle complex of pathogenic bacteria has many similarities with the bacterial flagella system.

In the research unit, we work to have a better understanding on the bacterial flagellum and on the T3SS. Bacterial motility and the function of the T3SS are linked to pathogenicity. In a time of antibiotic resistant bugs, understanding these systems at the fundamental level will help in a better design of therapeutic drugs.

1. Staff

• Dr. Fadel A. Samatey, Associate Professor
• Dr. Clive S. Barker, Researcher
• Dr. Yasuji Kido, Researcher
• Dr. Hideyuki Matsunami, Researcher
• Dr. Young-Ho Yoon, Researcher
• Dr. Paula Bulieris. Technician
• Irina Meshcheryakova, Technician
• Seiya Kitanobo, Techncian
• Tomoharu Inoue, Technician
• Dr. Yuzuki Manabe, Technician
• Saeko Hedo, Research Administrator

2. Collaborations

• Theme: Expression and purification of the membrane protein complexes
  • Type of collaboration: Joint research
  • Researchers:
    • Professor Keiichi Namba, Graduate School of Frontier Bioscience, Osaka University, Japan
    • Professor Katsumi Imada, Department of Macromolecular Science, Graduate School of Science, Osaka University, Japan

• Theme: Molecular dynamic of the bacteria flagellum by neutron scattering
  • Type of collaboration: Collaboration
  • Researchers:
    • Dr. Giuseppe Zaccaï, Institute of Laue-Langevin, Grenoble, France

• Theme: Structural Study of the bacterial Type II secretion system
  • Type of collaboration: Joint research
  • Researchers:
    • Dr.  Jean-Rome Voulhoux, CNRS, Institute of Structural Biology and Microbiology, Marseille, France

• Theme: Structural investigation of disordered proteins
  • Type of collaboration: Joint research
  • Researchers:
    • Professor Alla Kostyukova, School of Chemical Engineering and Bioengineering, Washington State University, Pullman, U.S.A.

• Theme: In vitro, in vivo high-yield production of membrane proteins
  • Type of collaboration: Collaboration
  • Researchers:
    • Dr. Bruno Miroux, Institut de Biologie Physico-Chimique, CNRS, Paris, France
    • Dr. Jean-Luc Popot, Institut de Biologie Physico-Chimique, CNRS, Paris, France
    • Dr. Francesca Zito, Institut de Biologie Physico-Chimique, CNRS, Paris, France

3. Activities and Findings

Within the trans-membrane trafficking unit we investigate how proteins are exported across the bacterial cell membrane. Our focus is the bacterial flagellum, which is used for motility and is the most complicated structure within the cell. The flagellum is related to the Type III secretion injectisomes produced by many types of pathogenic bacteria. The flagellum and the injectisomes harness the energy from the proton-motive force across the cell membrane to export proteins. The mechanism of function of the flagellum and injectisomes is an important area of biological research.

We recently investigated the connection between the activity of the respiratory chain, which builds the proton-motive force, and the assembly of the flagellum (1). The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localised to genes for subunits of respiratory complex I (NADH:quinone oxidoreductase-1). Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of respiratory complex I, and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain.

Respiration was rescued and the suppressed mutant strains grew better in Luria-Bertani broth medium and could use L-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterised by reversed-phase high performance liquid chromatography. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased respiratory complex I levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Thus, under certain growth conditions the suppressor mutations improve electron flow activity of respiratory complex I for cells bearing a ubiA deletion mutation, which in turn increased flagellar biogenesis and motility. This result showed that activity of respiratory complex I is important for assembly of the bacterial flagellum.

 

4. Publications

4.1 Journals 

• Meshcheryakov, V. A.,Kitao, A., Matsunami, H., Samatey, F. A. Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins. Acta Crystallogr D Biol Crystallogr, Pt 5, 69, P812-20 (2013)

• Meshcheryakov, V. A., Barker, C. S., Kostyukova, A. S., Samatey, F. A. Function of FlhB, a membrane protein implicated in the bacterial flagellar type III secretion system. PLoS One, 8,7, e68384 (2013)

• Stadler, A. M., Unruh, T., Namba, K., Samatey, F., Zaccai, G. Correlation between supercoiling and conformational motions of the bacterial flagellar filament. Biophys J, 105, 9, 2157-65 (2013)

• Galeva, A., Moroz, N., Yoon, Y. H., Hughes, K. T., Samatey, F. A., Kostyukova, A. S. Bacterial Flagellin-Specific Chaperone FliS Interacts with Anti-Sigma Factor FlgM. J Bacteriol, 196, 6, 1215-21 (2014)

 

4.2 Oral and Poster Presentations

• Samatey, Fadel A., Structural studies of membrane proteins: hopes and despairs, 43 years of wrestling with membrane proteins, IBPC, Paris France, October 25, 2013.

• Samatey, Fadel A., Nanoparticles as selective anti-bacterial compounds?, MINATEC Les midis, Grenoble, France, October 18, 2013.

• Barker Clive S., Post-transcriptional and post-translational control of the flagellar regulon rescues motility of a Salmonella enterica type III export fliO mutant, Biophysical Society 58th Annual meeting, San Francosco, USA, February 17, 2014.

• Barker Clive S., A minimized flagellar export apparatus without FliO, Flagellar Meeting , Hiroshima, Japan, March 2, 2014.

 

5. Meetings and Events

5.1 Research Visit

• Date: April 26, 2013 - May 5, 2013
• Venue: Lab1, Campus
•  Prof. Eric Martz, University of Massachusetts, Amhest, USA

• Date: September 30, 2013 - October 3, 2013
• Venue: Lab1, Campus
• Dr. Christine Ebel, Institut de Biologie Structurale CEA-CNRS-Universite Grenoble Alpes, France

• Date: December 1, 2013 - December 16, 2013
• Venue: Lab1, Campus
• Prof. Simon Silver, University of Illinois, Chicago, USA

• Date: February 22, 2014 - February 27, 2014
• Venue: Lab1, Campus
• Prof. Kelly Hughes, University of Utah, USA

• Date: March 10, 2014 - March 15, 2014
• Venue: Lab1, Campus
• Dr. Araine Briegel, California Institute of Technology, USA

 

5.2 Seminar

Title: AUC and SANS for structural studies

• Date: October 1, 2013
• Venue: Meeting Room C016 on Level C, Lab1
• Co-organizers: 
• Speakers: Christine Ebel, Ph.D
• Senior Scientist, Insititut de Biologie Structurale (IBS) SNRS, CEA, Universite Grenoble Alpes, France

 

Title: What is a virus? New world of viruses with megaviruses, pandoravirus and virophage

• Date: December 11, 2013
• Venue: Meeting Room C016 on Level C, Lab1
• Co-organizers: 
• Speakers: Prof. Simon Silver
• Professor, Department of Microbiology and Immunology, University of Illinois, Chicago, USA

 

Title: On the Higher Order Nature of the Genetic Code for Proteins

• Date: February 25, 2014
• Venue: Meeting Room C016 on Level C, Lab1
• Co-organizers: 
• Speakers: Prof. Kelly Hughes
• Professor, Department of Biology, University of Utah, USA

 

Title: Insights into the structure, assembly and function of chemoreceptor arrays by Electron Cryotomography

• Date: March 12, 2014
• Venue: Meeting Room C016 on Level C, Lab1
• Co-organizers: 
• Speakers: Araine Briegel, Ph.D
• Senior Scientist, Division of Biology, California Insititue of Technology, USA