Lipopolysaccharide is transported to the cell surface by a membrane-to-membrane protein bridge.

Gram-negative bacteria have an outer membrane that serves as a barrier to noxious agents in the environment. This protective function is dependent on lipopolysaccharide, a large glycolipid located in the outer leaflet of the outer membrane. Lipopolysaccharide is synthesized at the cytoplasmic membrane and must be transported to the cell surface. To understand this transport process, we reconstituted membrane-to-membrane movement of lipopolysaccharide by incorporating purified inner and outer membrane transport complexes into separate proteoliposomes. Transport involved stable association between the inner and outer membrane proteoliposomes. Our results support a model in which lipopolysaccharide molecules are pushed one after the other in a PEZ dispenser-like manner across a protein bridge that connects the inner and outer membranes.

LptE binds to and alters the physical state of LPS to catalyze its assembly at the cell surface.

The assembly of lipopolysaccharide (LPS) on the surface of Gram-negative bacterial cells is essential for their viability and is achieved by the seven-protein LPS transport (Lpt) pathway. The outer membrane (OM) lipoprotein LptE and the ß-barrel membrane protein LptD form a complex that assembles LPS into the outer leaflet of the OM. We report a crystal structure of the Escherichia coli OM lipoprotein LptE at 2.34 Å. The structure reveals homology to eukaryotic LPS-binding proteins and allowed for the prediction of an LPS-binding site, which was confirmed by genetic and biophysical experiments. Specific point mutations at this site lead to defects in OM biogenesis. We show that wild-type LptE disrupts LPS-LPS interactions in vitro and that these mutations decrease the ability of LptE to disaggregate LPS. Transmission electron microscopic imaging shows that LptE can disrupt LPS aggregates even at substoichiometric concentrations. We propose a model in which LptE functions as an LPS transfer protein in the OM translocon by disaggregating LPS during transport to allow for its insertion into the OM.

Origins of the regioselectivities in the Diels-Alder reactions of vinylindenes with 1,4-quinone monoketal and acrolein dienophiles.

Computations with density functional theory (B3LYP/6-31G(d)) have elucidated the origins of regioselectivities in the Diels-Alder reaction of vinylindene with a 1,4-quinone monoketal reaction that was employed as the key step in the synthesis of fluostatin C. Frontier Molecular Orbital theory and an electrostatic model are applied to the reactions of alkyl-substituted and vinylindene dienes with 1,4-quinone monoketal and acrolein dienophiles. Regiochemical results that deviate from expectation are explained by a progression from electronic to steric control upon the addition of a Lewis acid catalyst.

Pumping fluids in microfluidic systems using the elastic deformation of poly(dimethylsiloxane).

This paper demonstrates a methodology for storing and pumping fluids that provide a useful capability for microfluidic devices. It uses microfluidic screw valves to isolate fluids in poly(dimethylsiloxane) (PDMS) microcompartments, in which the pressure of the liquid is stored in the elastic deformation of the walls and ceiling of the compartments. Fluids can be stored under pressure in these structures for months. When the valves are opened, the walls and ceiling push the fluid out of the compartments into microfluidic channels. The system has five useful characteristics: (i) it is made using soft lithographic techniques; (ii) it allows multiple reagents to be preloaded in devices and stored under pressure without any additional user intervention; (iii) it makes it possible to meter out fluids in devices, and to control rates of flow of fluids; (iv) it prevents the user from exposure to potentially toxic reagents; and (v) it is hand-operated and does not require additional equipment or resources.