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     Molecular Genetics


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


  abstract

 

Our unit studies molecular aspects of two fundamental sets of processes in bacteria: transcription regulation and protein traffic. In particular, we are interested in how the transcription activator MalT responds to the presence of maltodextrins in the growth medium to turn on the expression of genes involved in their transport and metabolism. We are also exploring in the molecular mechanisms by which proteins are targeted to different cell compartments, are assembled into surface organelles or are secreted into the growth medium.



  report

cale

The Molecular Genetics Unit is composed of two subgroups, both of which study molecular aspects of fundamental life processes in Gram-negative bacteria: protein secretion and membrane biogenesis (Secretion group) and transcription activation (Regulation group).

Secretion group

One of the major projects in the secretion group for the last 15 years has been the characterization of the pathway by which the Gram-negative bacterium Klebsiella oxytoca secretes the amylolytic enzyme pullulanase. Recently, we have concentrated our efforts on the structural and functional aspects of two critical components of this secretion machinery, the secreton. One of these is the channel by which pullulanase is presumed to cross the outer membrane. The protein that probably constitutes the major part of this channel, protein, called secretin or PulD, forms a dodecameric complex that resembles a barrel that is apparently surrounded by 12 subunits of the other outer membrane protein, PulS. The latter, also called pilotin, is necessary for the membrane insertion and stability of PulD. The first three-dimensional structure of the secretin-pilotin complex was obtained by cryoelectron microscopy several years ago. In collaboration with Andreas Engel's group in Basel, we have recently improved several technical aspects of the particle analysis that will soon provide higher-resolution structures (see Figure 1 for an example). We are currently exploring different methods to visualize and identify domains of the secretin with the specific aims of determining which parts of the protein are involved in plugging the channel when it is not operational and where the pilotin is located.

We have proposed that some of the 13 components of the secreton form a piston-like structure (the pseudopilus) that pushes secreted proteins through the central channel of the secretin-pilotin barrel. This hypothetical piston-like structure is the second of the two critical components of the secreton that we are examining. When the gene coding for the major constituent (PulG) of the pseudopilus is overexpressed, pili appear on the surface of the bacteria. These pili have been purified and examined by electron microscopy and PulG has been purified and crystallized (Figure 2). Model building is currently being used to generate a three-dimensional image of how PulG packs into the filament.

We also have an almost equally long-standing interest in the way lipoproteins are sorted to different compartments in the bacterial cell envelope. Lipoproteins carry a fatty acyl motif at their N-terminal end that anchors them in the cell envelope. In Gram-negative bacteria, the attachment site is usually in the periplasmic face of either the inner (plasma) or outer membrane. We and others have shown that a lipoprotein can be switched from one location to the other by changing the amino acid at position 2 in the sequence. We are currently exploring the molecular mechanisms that underlie this sorting phenomenon, and in particular whether the amino acid at position 2 influences the nature or the extent of fatty acylation of the cysteine residue at position 1 of the lipoprotein.

Regulation group

Our work is based on the maltose system of the Enterobacteriaceae. These bacteria transport and metabolize maltose via a number of proteins whose genes form the maltose regulon and are controlled by a specific transcription activator called MalT. MalT is the best-characterized member of a new family of large bacterial transcription activators.

MalT comprises 4 structural domains. The first three, which are specific to proteins in the MalT family, seem to define a new signal transduction module that alters the ability of the fourth domain to bind to DNA and interact with RNA polymerase through its capacity to undergo controlled oligomerisation. We are presently exploring numerous distinctive aspects of the way this protein functions. Firstly, the high number of signals that control MalT activity raises the question of how these signals are integrated by the protein in order to provide the appropriate level of transcription activation. Indeed MalT only oligomerises in the presence of ATP and maltotriose (the inducer). In addition, MalT activity is also negatively controlled by three proteins, MalK, MalY and Aes. Although the three proteins act independently, they exert the same effect, which is to prevent the binding of maltotriose. Secondly, we are examining the hypothesis that the ATPase activity of MalT, a property rarely associated with transcription activators that act together with RNA polymerase containing the major sigma factor, influences the competition between maltotriose and the negative effectors. The final distinctive

property of MalT that we are exploring is its mode of multimerisation, which allows it to recognise quite different arrangements of its specific binding sites in the promoters it controls. Our mechanistic studies of these phenomena exploit both genetic and biochemical methodologies, as well as structural approaches (X-ray crystallography of the complete protein or domains thereof and cryoelectron microscopy).

Figure 1. Crystal structure of PulG. The presence of antiparallel beta-strands is typical of type IV pilins, to which PulG is related (Photograph courtesy of Karsten Shäffer and Wolfram Welte, University of Konstanz, Germany)

Figure 2. Reconstitution of PulD particles examined by electron microscopy (Photograph by courtesy of Mohamed Chami and Andreas Engel, University of Basel, Switzerland).

Keywords: Gram-negative bacteria, secretion, outer membrane, lipoprotein maltose, transcription activation



  web site

puce More informations on our web site


  publications

puce Publications 2003 of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Lavenir, Armelle, Secretary, alavenir@pasteur.fr Evelyne Richet, CNRS, Research Director,erichet@pasteur.fr

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

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

Nicolas Bayan, University of Paris XI, Assistant Professor, nbayan@pasteur.fr
Rolf Koehler, postdoc EC, rkoehler@pasteur.fr

Guillaume Vignon, PhD student, gvignon@pasteur.fr

Carine Robichon, PhD student, crobicho@pasteur.fr

Nicolas Joly, PhD student, jolyn@pasteur.fr
Dominique Vidal-Ingigliardi, Engineer, dvidalin@pasteur.fr

Ingrid Guilvout, Senior Technician, ingvout@pasteur.fr

Nathalie Nadeau, Technician, nnadeau@pasteur.fr

Maria Reyngoud, Laboratory Assistant

Activity Reports 2003 - Institut Pasteur
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