Molecular Genetics  

  HEADProf. PUGSLEY, Anthony /
  MEMBERSDr. FRANCETIC, Olivera / Prof. BAYAN, Nicolas (10%) / Dr. BUDDELMEIJER, Nienke / Dr. KREHENBRINK, Martin / Dr. RICHET, Evelyne / Dr. DANOT, Olivier / GUILVOUT, Ingrid / VIDAL-INGIGLIARDI, Dominique
Dr. KOLB, Annie / Dr. NOREL, Françoise / NADEAU, Nathalie / ROBBE-SAULE, Véronique / LAVENIR, Armelle / REYNGOUD, Maria / LEGAT, Geneviève / COLIN, Séverine / CAMPOS, Manuel / MARQUENET, Emélie / BERAUD, Mélanie

  Annual Report

This unit is composed of three groups headed by Dr. Kolb, Dr. Richet and Prof. Pugsley. Their common aim is to understand the molecular details of transcription regulation, signal transduction and protein traffic in bacteria by protein biochemistry and structural biology, biophysics and classical and molecular genetics.

Drs Kolb and Norel mainly study the RNA polymerase stationary phase sigma factor S, the way it recognizes and binds to target DNA in its specific promoters, and the way its assembly into RNA polymerase is controlled and responds to growth phase and other factors. Proteins that modulate the association between sigma S and core polymerase are being identified and characterized, and aspects of differential regulation of stationary phase gene expression explored in Salmonella.

Dr. Richet and Dr. Danot work on a transcription activator called MalT, which responds to the presence of maltodextrins to stimulate the expression of genes required for their transport and metabolism in E. coli. MalT, the best-characterized member of an important group of signal-transducing ATPases (STAND), is a large (>100 kDa) protein composed of four domains responsible for DNA binding, ligand recognition, ATP hydrolysis and multimerization. MalT activation involves maltotriose binding and multimerization that allow it to bind to specific sequences in its target promoters. The crystal structure of part of the ligand-binding domain of the E. coli MalT revealed potential maltotriose-binding site that was mapped by mutagenesis. Recent studies revealing the role of ATP hydrolysis in resetting the switch mechanism from the active to the inactive state open up new avenues to study conformational changes in other STAND ATPases.

The third group works on protein traffic and membrane assembly. Its members concentrate mainly on the bacterial type II secretion system, of which bthree out of its 12 protein components are currently investigated. Dr. Pugsley works on secretin, which forms a dodecameric channel in the outer membrane to permit folded proteins to exit the cell from the periplasm. Understanding the structure-function aspects of this protein is one of the major goals of the group. The factors necessary for folding, multimerization and specific outer membrane insertion of this protein are being prioritized. These processes involve a specific, apparently multifunctional chaperone that is also being characterized. Dr. Francetic works on the third protein of particular interest. It forms a filament that resembles surface appendages called pili. However, the “pseudopilus” normally exists within the periplasm, where it could promote protein secretion by cycles of assembly and disassembly that literally push the secreted protein through the secretin channel by displacing the plug at its center. To test this model, we are refining the structure of the protein and of the filament it forms and testing aspects of subunit interaction and dynamics by site directed mutagenesis. Finally, this group is also interested in how this unique secretion system achieves exquisite selectivity (only one protein is secreted out of >150 proteins in the periplasm). We are currently identifying residues in the secreted protein that are required for its recognition and the components of the secretion machinery that act as its receptors.

Within the same group, Dr. Buddelmeijer works on the enzymes that link the fatty acids onto the 100–or–so lipoproteins made by E. coli. They have very interesting catalytic actions, are essential, and have surface-exposed catalytic sites that make them potential targets for antibacterial agents specific for Gram-negative bacteria. This important research area was recognized as a target for high priority funding by the EU and is one that we feel deserves greater support within the Institut Pasteur.

Keywords: Protein secretion, lipoprotein, RNA polymerase, sigma factor, STAND ATPase, transcription factor


Assembly and insertion of PulD secretin C-domain into liposomes during and in vitro transcription-translation reaction. Liposomes harvested from the reaction contain abundant PulD secretin complexes densely packed in the lipid phase (unpublished).


Marquenet, E., and E. Richet. 2007. How integration of positive and negative regulatory signals by a STAND signaling protein depends on ATP hydrolysis. Mol Cell 28: 187-199.

Mouratou, B., F. Schaeffer, I. Guilvout, D. Tello-Manigne, A. P. Pugsley, P. M. Alzari, and F. Pecorari. 2007. Remodeling a DNA binding protein as a specific in vivo inhibitor of bacterial secretin PulD. Proc. Natl Acad. Sci. USA. 104: 17983-17988.

Collin, S., I. Guilvout, M. Chami, and A. P. Pugsley. 2007. YaeT-independent multimerization and outer membrane association of secretin PulD. Mol Microbiol 64:1350-7.

Guilvout, I., M. Chami, A. Engel, A. P. Pugsley, and N. Bayan. 2006. Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin. EMBO J 25:5241-5249.

Robbe-Saule, V., M. D. Lopes, A. Kolb, and F. Norel. 2007. Physiological Effects of Crl in Salmonella Are Modulated by {sigma}S Level and Promoter Specificity. J Bacteriol 189:2976-87.

Activity Reports 2007 - Institut Pasteur
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