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     Molecular Genetics - CNRS URA 2172


  Director : Pugsley, Anthony P (max@pasteur.fr)


  abstract

 

The Molecular Genetics Unit studies fundamental aspects of bacterial gene regulation, protein targeting and envelope assembly using a wide variety of techniques ranging from genetics and molecular biology through to cell biology, biochemistry, biophysics and structural biology, mainly in Escherichia coli. Our central objective is to provide refined molecular information on the structure and dynamics of cellular components involved in these processes.



  report

cale

The advantages of Escherichia coli and related bacteria as model organisms for studying fundamental aspects of life processes such as gene expression and cell assembly and organization are numerous and irrefutable. We have exploited these advantages to provide greater understanding of protein traffic and of signal perception and transduction to genetic regulatory circuits. Our studies have established principles of general interest and have had substantial impact beyond the boundaries of microbiology.

Much of our work on protein traffic involves attempts to unravel the molecular complexities of the type II secretion system, which is found in most species of Gram-negative bacteria. Type II secretion occurs in two steps. In the first, exoproteins are transported across the inner membrane, usually by the general (Sec) export pathway. They then fold and engage the dedicated second step that involves 12 essential components required for substrate recognition (only one or a limited number of exoproteins is secreted), and energized transport across the outer membrane. Essential components of this pathway include a potentially dynamic, periplasmic, pilus-like structure (the pseudopilus) that probably acts as a piston to push exoproteins through the outer membrane, a tightly gated channel (the secretin) in the outer membrane though which the exoproteins traverse this membrane, a pilot protein (pilotin) needed to insert the secretin in this membrane and an energized motor that is probably required for pseudopilus dynamics.

We have concentrated our efforts on the first three of these factors. We have shown that secretin is a dodecameric protein complex that is embedded in the outer membrane with a substantial periplasmic domain. Limited proteolysis removes a large part of this periplasmic domain to leave a core dodecameric complex. This core domain acts as an autonomous unit capable of forming correctly organized, membrane-inserted dodecamers. Pilotin is needed to direct the complex to the outer membrane (in its absence, the complex associates with the inner membrane) but the kinetics of pilotin-secretin association, traffic through the periplasm and complex formation still need to be sorted out. Attempts are being made to analyse the ability of the core domain to oligomerize and insert into lipid bilayers in vitro. The protein will be analysed by 2D- and 3D-crystallography. The separate N domain exists as a stable monomer in solution. X-ray crystallography will be used to determine its 3D structure. The structure of pilotin together with the secretin domain to which it binds will also be determined. We are confident that these studies will provide a high resolution image of the complete secretin-pilotin complex.

The structure of the major component of the pseudopilus, pseudopilin G, has already been determined. The structure lacks the N-terminal hydrophobic region needed for its export, its insertion into the inner membrane (where it forms a pool of unassembled protomers) and its assembly. The way in which this protein is targeted to the inner membrane remains to be determined. We are currently creating gene fusions and mutations that affect the signal-anchor sequence, and studying mutations affecting the Sec and associated signal recognition particle pathways to analyse their effects on pseudopilin G export, assembly and function.

Fluorescent reporter proteins have recently been used to great effect to study other aspects of secreton function. For example, fluorescence microscopy of green fluorescent derivatives of secreton components indicates that they are located in discrete patches throughout the inner membrane. Red fluorescent proteins were used to investigate the regions in an exoprotein that are needed for efficient secretion. These and other studies led to the identification of a crucial region near the centre of the polypeptide that might be recognized by specific secreton components. The 3D crystal structure, which is currently being refined, should allow us to identify key residues involved in this interaction and to employ mutagenesis and allele-specific suppression analyses to identify critical residues in interacting components.

Red fluorescent proteins were also recently used to study how lipoproteins are targeted to specific sites in E. coli cells. In situ localization by fluorescence microscopy of plasmolysed cells allowed the inner and outer membranes to be clearly visualized. Red fluorescent lipoproteins were found to localize correctly according to the sorting rules we established previously. Furthermore, the same sorting rules were shown to be perfectly conserved in a panel of gamma-proteobacteria.

Lipoprotein processing and modification in Gram-negative bacteria involves three enzymes that are essential for viability. One of these, apolipoprotein N-acyltransferase, received particular attention because of its key role in lipoprotein maturation and sorting to the correct cell compartment. We have now begun a genetic, biochemical and structural analysis of this enzyme in order to understand its catalytic activity and to search for inhibitors that might open the way for the rational design of compounds with antibacterial activity.

Our studies on signal perception and transduction stem from our long-standing interest in the maltose system from E. coli. The master regulator of the maltose regulon is MalT, a transcription factor that turns out to be the prototype of a new class of signalling ATPases, the STAND (signal transduction ATPases with numerous domains) proteins. These widespread proteins function as hubs that integrate multiple regulatory signals and that, in response, either transmit a signal or execute a task. Because of their structural complexity, very few STAND ATPases have been studied in vitro. As a result, the underlying mechanisms remain unclear. The MalT protein is in equilibrium between a monomeric, inactive form and a multimeric, transcriptionally active form. The transition between the two forms is controlled by three negative effectors (the proteins MalK, MalY and Aes) and by a positive effector (maltotriose). The three N-terminal MalT domains: DT1 (ATPase domain) and DT2, which both characterize the STAND class, and DT3, a sensing domain, constitute a signal integration module that responds to incoming signals via a change in its quaternary structure, thereby altering the ability of DT4, the DNA-binding domain of MalT, to bind cooperatively to the array of MalT sites present in the target promoters. Our aim is to elucidate (a) the mechanisms whereby MalT responds to incoming signals, and (b) the role of the ATPase activity of MalT in protein function.

Photos :

Photo 1: 3D structure of secretin PulD determined by cryoelectron microscopy.

Photo 2: Localization of a green fluorescent protein-tagged type II secretion motor component by epifluorescene microscopy.

Keywords: protein secretion, outer membrane, protein channels, signal transduction, transcription



  publications

puce Publications 2005 of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Lavenir, Armelle, Institut Pasteur, (Secretary, alavenir@pasteur.fr) Bayan, Nicolas, University of Paris XI, (Professor, nbayan@pasteur.fr)

Danot, Olivier, Institut Pasteur, (Staff Scientist, olivdano@pasteur.fr)

Francetic, Olivera, Institut Pasteur, (Staff Scientist, ofrancet@pasteur.fr)

Richet, Evelyne, CNRS, (Research Director, erichet@pasteur.fr)

Buddelmeijer, Nienke, Postdoc

Lewenza, Shawn, Postdoc

Marquenet, Emélie, PhD

Collin, Séverine, Masters II

Guilvout, Ingrid, (Engineer, ingvout@pasteur.fr)

Nadeau, Nathalie, (Technician, nnadeau@pasteur.fr)

Reyngoud, Maria, (Laboratory Assistant)

Vidal-Ingigliardi, Dominique, (Engineer, dvidalin@pasteur.fr)


Activity Reports 2005 - Institut Pasteur
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