Unit: Molecular Genetics - CNRS URA 2172
Director: Pugsley, Anthony P
The activities of the Molecular Genetics Unit concentrate on the quest for a deeper molecular understanding of two fundamental life processes; the way specific stimuli lead to the activation of transcription at promoters that respond to them and the way proteins are localized to specific cell compartment and how they assemble into and function as macromolecular machines. Our studies, which are conducted mainly in the bacterium Escherichia coli, have broad relevance to several scientific disciplines, including structural and cell biology.
At a time when increasing attention is being devoted to more general and integrated aspects of biology through studies grouped together as Systems Biology, it is vital to reinforce efforts to achieve a deeper molecular understanding of fundamental processes on which all biological systems are based. These considerations motivate us to pursue our molecular investigation of two such processes, signal perception and transduction to genetic regulatory circuits and protein targeting. We have chosen to study these processes in the bacterium Escherichia coli, which offers many advantages compared to more complex experimental systems. Nevertheless the impact of our studies spreads well beyond the boundaries of microbiology.
Our studies on signal perception and transduction stem from our long-standing interest in maltodextrin metabolism by E. coli. The central regulatory element in this system, the protein MalT, is a positive transcription factor that is specifically activated by maltotriose, which promotes its binding to dedicated sites in the promoters of the operons it controls. We have shown that this switch to the activated state coincides with the multimerization of MalT. Oligomerization is specifically impeded by any of other three negative effectors proteins, one of which, MalK, is a peripheral membrane component of the maltodextrin permease in the bacterial plasma membrane. MalK and maltotriose compete for binding to MalT. Our current studies are aimed at defining the mechanisms by which the molecular switch is controlled. We have already identified sites in MalT to which maltotriose and MalK bind and the structural transitions that these events induce. We have also begun to investigate the role of the ATPase activity of MalT in this process of signal integration. Further studies of MalT will reveal important insights into the mechanisms of signal transmission in the newly recognized STAND family of ATPases, of which MalT is the prototype.
Our recent work on protein traffic stems from our early observation that production and secretion the amylolytic enzyme pullulanase by the close E. coli relative Klebsiella oxytoca is regulated by MalT. The two-step secretion of this enzyme requires both the universal Sec system in the bacterial plasma membrane and a dedicated secretion machinery, the secreton, composed of 13 different proteins, the expression of which is MalT-dependent. A detailed structural investigation has been undertaken in order to understand how the secreton drives pullulanase translocation across the outer membrane. Two secreton components have received particular attention. The recently obtained cryoelectron microscopy structure of one of them, the secretin (protein D), indicates that it probably forms a gated channel through which the enzyme crosses the outer membrane. Combined crystallographic and electron microscopic analyses of the other characterized secreton component, the pseudopilin G, indicate that it assembles into a pilus-like structure that we propose is the dynamic core of a piston-like motor that pushes pullulanase through the secretin channel. Structural and conceptual models based on these data are now being tested by site-directed mutagenesis and refined mapping of subdomains. Finally, we have mapped several regions of the pullulanase polypeptide that appear to be essential for its secretion. X-ray crystallographic analysis of the enzyme that is now under way will allow us to pinpoint these regions and to undertake refined mapping of potential sites involved in its docking with different secreton components.
We are also interested in the assembly of the complex secreton machinery, and particularly whether it is located at specific sites in the cell and the order in which the components are brought together. Recent data indicate that chimeras comprising red or green fluorescent reporter proteins fused to different secreton components are functional and are assembled at discrete locations in the cell envelope. These studies will lead us into the analysis of the molecular interactions between- and dynamics of the secreton machinery components and thence to investigate broader aspects of bacterial cell organization.
The secreton is one of several distinct protein translocation machineries found in bacteria. Many of them are well characterized but very little is known about others, particularly in bacteria with unusual envelope structures. One such bacterium is Corynebacterium glutamicum, which possesses a highly impermeable pseudo-outer membrane composed mainly of mycolic acid and through which this bacterium translocates a protein that assembles into a coat on the cell surface. How does this protein cross the pseudo-outer membrane? To answer this question, we have started to analyse two mutants that exhibit decreased levels of surface protein secretion. The characterization of the genes that are mutated represents the first step towards the identification of a potentially novel translocation machinery that has broad implications for the secretion of virulence factors by closely related pathogenic bacteria such as Mycobacterium tuberculosis.
The molecular genetics unit has a long-standing interest in lipoproteins, a unique class of exported proteins with a fatty acylated N-terminus that anchors them in the cell envelope. Our recent studies of the way lipoproteins are sorted to the cell surface, the inner face of the outer membrane or the outer face of the inner membrane have now led us to examine the enzymes responsible for lipoprotein fatty acylation and proteolytic processing. These enzymes are essential for viability in E. coli and Salmonella enterica but, as with the mechanisms of lipoprotein sorting to different cell envelope compartments, very little is known about their role in other bacteria. We have recently started to investigate this question in a study motivated, in part, by the fact that enzymes involved in cell envelope biogenesis are ideal targets for the development of novel antimicrobial agents. Absence of the enzyme responsible for the addition of the third and final fatty acid causes outer membrane lipoproteins to remain in the inner membrane, leading to severe envelope disorganization. We are currently mapping sites in the enzymes that are essential for its function as a prelude to a fuller structure-function analysis that will reveal the different steps involved in the fatty acid transfer reaction.
Structure of PulG, a component of the K. oxytoca secretion and of the pilus that it forms
Keywords: signal transduction, transcription activation, secretion, lipoproteins