Welcome to the Silhavy Lab
Membrane biogenesis and signal transduction
All cells have subcellular compartments that are bound by lipid bilayers, and this three-dimensional organization is essential for life. If we consider lipid bilayers themselves as compartments, then Gram-negative bacteria, such as Escherichia coli, have four distinct subcellular locations: the cytoplasm, inner membrane (IM), periplasm, and outer membrane (OM). The noncytoplasmic compartments are collectively termed the cell envelope. We wish to understand cellular assembly, in particular, the process of OM biogenesis. The OM is an asymmetric lipid bilayer containing phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. Membrane spanning outer membrane proteins (OMPs) typically assume a beta-barrel conformation. All OM components are synthesized in the cytoplasm or the IM and therefore, OM biogenesis requires the transport of these molecules across the cell envelope for assembly at their final cellular location. Strikingly, these transport and assembly processes occur in an environment that lacks an obvious energy source such as ATP. We have used a combination of genetics, biochemistry, and bioinformatics to identify the cellular machinery required for the assembly of OMPs and LPS in the OM. Both of these assembly machineries have protein components in every cellular compartment, and this spatial organization suggests the temporal order of component function. Current effort in the lab is directed towards understanding how these machines function in molecular terms, and how the various envelope stress responses maintain cell integrity.
When E. coli deplete their environment of essential nutrients they enter a physiological state termed stationary phase in order to survive prolonged starvation and resist various toxic insults. Entry into stationary phase is accompanied by large changes in gene expression, most of which are mediated by sigma-S, the central regulator of the stationary phase response. Sigma-S regulation is complex and occurs at all levels, but regulated proteolysis is of primary importance for carbon starvation. In log phase cells, the two-component response regulator SprE directs sigma-S to the ClpP/ClpX protease, but as cells enter stationary phase this destruction ceases and sigma-S accumulates. SprE belongs to the response regulator family of two-component regulatory proteins. As such it was assumed that the activity of SprE was controlled by phosphorylation. We have shown that this model is not correct. Rather icellular ATP levels directly control sigma-S stability. We have also shown that SprE has a second function in regulating mRNA stability and current efforts in the lab are directed towards understanding this regulatory mechanism.