Analysis of surface protein expression reveals the growth pattern of the gram-negative outer membrane.

The outer membrane (OM) of Gram-negative bacteria is a complex bilayer composed of proteins, phospholipids, lipoproteins, and lipopolysaccharides. Despite recent advances revealing the molecular pathways underlying protein and lipopolysaccharide incorporation into the OM, the spatial distribution and dynamic regulation of these processes remain poorly understood. Here, we used sequence-specific fluorescent labeling to map the incorporation patterns of an OM-porin protein, LamB, by labeling proteins only after epitope exposure on the cell surface. Newly synthesized LamB appeared in discrete puncta, rather than evenly distributed over the cell surface. Further growth of bacteria after labeling resulted in divergence of labeled LamB puncta, consistent with a spatial pattern of OM growth in which new, unlabeled material was also inserted in patches. At the poles, puncta remained relatively stationary through several rounds of division, a salient characteristic of the OM protein population as a whole. We propose a biophysical model of growth in which patches of new OM material are added in discrete bursts that evolve in time according to Stokes flow and are randomly distributed over the cell surface. Simulations based on this model demonstrate that our experimental observations are consistent with a bursty insertion pattern without spatial bias across the cylindrical cell surface, with approximately one burst of ≈ 10(-2) µm(2) of OM material per two minutes per µm(2). Growth by insertion of discrete patches suggests that stochasticity plays a major role in patterning and material organization in the OM.

Controlling DNA capture and propagation through artificial nanopores.

Electrophorescing biopolymers across nanopores modulates the ionic current through the pore, revealing the polymer's diameter, length, and conformation. The rapidity of polymer translocation ( approximately 30,000 bp/ms) in this geometry greatly limits the information that can be obtained for each base. Here we show that the translocation speed of lambda-DNA through artificial nanopores can be reduced using optical tweezers. DNAs coupled to optically trapped beads were presented to nanopores. DNAs initially placed up to several micrometers from the pore could be captured. Subsequently, the optical tweezers reduced translocation speeds to 150 bp/ms, about 200-fold slower than free DNA. Moreover, the optical tweezers allowed us to "floss" single polymers back and forth through the pore. The combination of controlled sample presentation, greatly slowed translocation speeds, and repeated electrophoresis of single DNAs removes several barriers to using artificial nanopores for sequencing, haplotyping, and characterization of protein-DNA interactions.