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


  abstract

 

We are studying molecular aspects of several fundamental life processes in Gram-negative bacteria, notably protein traffic and membrane biogenesis, and transcription activation. The model systems on which most of our work is based are the pullulanase secretion system of Klebsiella oxytoca, and MalT protein, the transcriptional activator of the maltose regulon in Escherichia coli.



  report

cale

The molecular Genetics Unit is composed of two subgroups, both of which are studying molecular aspects of fundamental life processes in Gram-negative bacteria: protein secretion and membrane biogenesis (Secretion group) and transcription activation (Regulation group). A large part of 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. In bacteria belonging to the genus Klebsiella, the synthesis and secretion of an amyloytic enzyme called pullulanase is also under MalT control. We are specifically interested in how pullulanase is secreted and how MalT activates transcription in response to the presence of maltodextrins in the growth medium.

Secretion group

The pullulanase secretion pathway is the archetype of the type II secretion pathway. This machinery, called the secreton, is composed of at least 13 specific proteins. Its role is to select specific proteins in the periplasm, the compartment located between the two lipid bilayer membranes of the Gram-negative bacteria cell envelope, and to transport them across the outer membrane. Among the 13 secreton components, only 2 are located in the outer membrane. Purification and analysis of one of them, the secretin PulD, revealed it to be a dodecameric complex that resembled a barrel that was apparently surrounded by 12 subunits of the other outer membrane secreton component, 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 cryoelectronmicroscopy several years ago. In collaboration with Andreas Engel's group in Basel, we have recently overcome several of the technical difficulties inherent in the application of this methodology to the analysis of purified membrane proteins. We have adopted a new technique in which histidine-tagged secretin-pilotin complexes are immobilized on a lipid monolayer in which the polar headgroups carry a nickel ion. The first images obtained by this technique (Figure 1) confirm the structure first determined by classical methods and encourage us to pursue its use to obtain higher resolution and to identify individual domains of PulD or PulS within the complex.

We have recently proposed that some of the secreton components with sequence similarities to the subunits of type IV pili might form a piston-like structure (the pseudopilus) that pushes secreted proteins through the central channel of the secretin-pilotin barrel. When the gene coding for the major constituent of the pseudopilus is overexpressed, pili appear on the surface of the bacteria (Figure 2). These pili can be harvested in sufficient quantities to permit low-resolution analysis of their structure by electronmicroscopy and the purification of sufficient quantities of pseudopilin subunits for crystallography and high resolution structural analysis by X-ray diffraction. By a combination of these two approaches, we should soon be in a position to propose a model of the pseudopilus on which to base further experiments to understand its function, structure and biogenesis.

Among the other projects currently pursued by members of the secretion group, we are interested in understanding the role of other secreton components, notably in recognition of the proteins to be secreted and in energy coupling pseudopilus assembly and secretion. We are also studying the secreton of E. coli, which we have shown to secrete a chitinase, with the aim of finding out how it is regulated, since it is not expressed under standard laboratory conditions. Finally, we are increasingly interested in the biogenesis and localization of lipoproteins in the bacterial cell envelope.

Regulation group

MalT is easily the best-characterized member of a new family of large bacterial transcription activators. MalT is almost completely different from any other model transcription activator studied so-far. Further studies of MalT will undoubtedly reveal many new and important aspects of the control of protein-DNA interactions and transcription.

We are currently exploring several unique aspects of MalT, most notably the multiple signals that control its activity, the aim being to determine how these different signals are integrated at the level of MalT protein structure. MalT is only active in the presence of ATP and maltotriose, its two positive effectors. ATP is not itself an inducer, its hitherto unexplored role being probably related to its hydrolysis by MalT. Three unrelated proteins, MalK, MalY and Aes, are able to act as negative effectors of MalT. MalK, the energy coupling ATPase component of the maltodextrin permease, probably couples the activity of the maltodextrin permease to activation of MalT and, hence expression of the maltose regulon.

A substantial part of our work is devoted to structural aspects of MalT function. We have found that the formation of an active transcription complex at a MalT-dependent promoter involves a series of tertiary and quaternary structure changes in the protein. MalT appears to be in equilibrium between active and inactive conformations, the first being stabilized by maltotriose and ATP, the second being induced by the negative effector proteins. Thus, the effectors control the switch between a minimum of two conformational states of MalT whose biophysical and structural analysis, currently underway, will lead to a better molecular understanding of how the effectors act. We have shown that the next step in the formation of an active transcription complex is MalT multimerization (see Figure 3), which probably facilitates co-operative binding to the MalT binding sites present in the target promoters. The fact that the organization of the MalT sites (MalT recognizes an asymmetrical binding motif) varies with the promoter suggests that MalT complexes differing in their molecular architecture are able to bind to different promoters.

Figure 1. Cryoelectronmicroscopy of PulD-PulS complexes immobilized on a lipid monolayer with nickel-substituted headgroups showing an averaged top view of the complex. The tentatively-identified PulS (pilotin) subunits are numbered 1 to 12. The inner ring is probably composed of 12 PulD (secretin) subunits. The material in the center of the ring probably represents a proteinaceous plug that blocks the channel formed by PulD. (Photograph by courtesy of Mohamed Chami and Henning Stahlberg, University of Basel, Switzerland).

Figure 2. Confocal microscopy of E. coli K-12 expressing the pul secreton genes carried by a multiple copy number plasmid. PulG pseudopili are labelled with fluorescent antibodies. Bacteria are stained red. Control bacteria lack the PulG pseudopilin and are unable to make pili.

Figure 3. Cryoelectronmicroscopy of complexes formed by MalT with bound ATP and maltotriose. The images are an averaged view of the complex and a model of one of the elements from which it appears to be formed. (Images by courtesy of Eric Larquet).

Keywords: Bacteria, Enterobacteriaceae, Secretion, Outer membrane, Maltose, Transcription activation



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  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

Odile Mary-Possot, Institut Pasteur, Staff Scientist,opossot@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


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