I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

This Review summarizes methods for constructing systems and structures at micron or submicron scales that have applications in microbiology. These tools make it possible to manipulate individual cells and their immediate extracellular environments and have the capability to transform the study of microbial physiology and behaviour. Because of their simplicity, low cost and use in microfabrication, we focus on the application of soft lithographic techniques to the study of microorganisms, and describe several key areas in microbiology in which the development of new microfabricated materials and tools can have a crucial role.

Herein we describe a versatile new strategy for producing monodisperse solid particles with sizes from 20 to 1000 mm. The method involves the formation of monodisperse liquid droplets by using a microfluidic device and shaping the droplets in a microchannel and then solidifying these drops in situ either by polymerizing a liquid monomer or by lowering the temperature of a liquid that sets thermally. This method has the following features: 1) It produces particles with an exceptionally narrow range of sizes.[1] 2) A new level of control over the shapes of the particles is offered.3) The mechanism for droplet formation allows the use of a wide variety of materials including gels, metals, polymers, and polymers doped with functional additives. 4) The procedure can be scaled up to produce large numbers of particles.

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier that defines cell shape and allows the cell to sustain large mechanical loads such as turgor pressure. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphology and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiology of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field.

The motion of peritrichously flagellated bacteria close to surfaces is relevant to understanding the early stages of biofilm formation and of pathogenic infection. This motion differs from the random-walk trajectories of cells in free solution. Individual Escherichia coli cells swim in clockwise, circular trajectories near planar glass surfaces. On a semi-solid agar substrate, cells differentiate into an elongated, hyperflagellated phenotype and migrate cooperatively over the surface, a phenomenon called swarming. We have developed a technique for observing isolated E. coli swarmer cells moving on an agar substrate and confined in shallow, oxidized poly(dimethylsiloxane) (PDMS) microchannels. Here we show that cells in these microchannels preferentially 'drive on the right', swimming preferentially along the right wall of the microchannel (viewed from behind the moving cell, with the agar on the bottom). We propose that when cells are confined between two interfaces--one an agar gel and the second PDMS--they swim closer to the agar surface than to the PDMS surface (and for much longer periods of time), leading to the preferential movement on the right of the microchannel. Thus, the choice of materials guides the motion of cells in microchannels.

Many proteins reside at the cell poles in rod-shaped bacteria. Several hypotheses have drawn a connection between protein localization and the large cell-wall curvature at the poles. One hypothesis has centered on the formation of microdomains of the lipid cardiolipin (CL), its localization to regions of high membrane curvature, and its interaction with membrane-associated proteins. A lack of experimental techniques has left this hypothesis unanswered. This paper describes a microtechnology-based technique for manipulating bacterial membrane curvature and quantitatively measuring its effect on the localization of CL and proteins in cells. We confined Escherichia coli spheroplasts in microchambers with defined shapes that were embossed into a layer of polymer and observed that the shape of the membrane deformed predictably to accommodate the walls of the microchambers. Combining this technique with epifluorescence microscopy and quantitative image analyses, we characterized the localization of CL microdomains in response to E. coli membrane curvature. CL microdomains localized to regions of high intrinsic negative curvature imposed by microchambers. We expressed a chimera of yellow fluorescent protein fused to the N-terminal region of MinD-a spatial determinant of E. coli division plane assembly-in spheroplasts and observed its colocalization with CL to regions of large, negative membrane curvature. Interestingly, the distribution of MinD was similar in spheroplasts derived from a CL synthase knockout strain. These studies demonstrate the curvature dependence of CL in membranes and test whether these structures participate in the localization of MinD to regions of negative curvature in cells.A central question in cell biology is how the spatial organization of proteins and lipids is established, maintained, and replicated and how it fluctuates in response to external stimuli. Eukaryotic cells use several mechanisms to accomplish this task, including a dynamic cytoskeleton that controls the spatial and temporal position of proteins, nucleic acid, and organelles (1). The formation of lipid microdomains in the membrane also is involved in the localization of integral membrane proteins (2). These mechanisms play a critical role in cell physiology and behavior.Bacteria also use mechanisms for controlling the intracellular location of proteins, lipids, and nucleic acid. The persistent historical view of bacterial cells as lacking spatial control over their intracellular components inhibited the field. Bacteria are sophisticated organisms that use tightly regulated physiological mechanisms similar to many of those used by eukaryotic cells, including controlling shape, regulating growth and division, transporting intracellular components (e.g., proteins, plasmids, DNA, and RNA), and polarizing the cell (3, 4). These organisms have several remarkable structural characteristics, but one of the most fundamental questions in this area of microbiology-how cells produce, maintain, and replicate their spatial organization-still...

through 20-gauge (nominal inner diameter: 0.6 mm) needles into rotating 40 wt.-% PEI (branched; average molecular weight, M w~2 5 000; water-free; Aldrich) solutions in methanol.The SWNT/PEI composite fibers were characterized by SEM (LEO 1590 VP microscope) and micro-Raman spectroscopy (Jobin± Yvon Horiba high-resolution LabRam micro-Raman spectrometer; helium±neon Spectra Physics laser, model 127, with excitation wavelength, k exc = 632.8 nm; resolution =~1 cm ±1 ). The mechanical properties of these fibers were measured at room temperature with an Instron MicroTester (using a 1 cm gauge length and a constant strain rate of 0.9±1.2 % min ±1. Four-probe electrical conductivities were obtained from resistance values measured using a Keithley 2000 Multimeter and using the fiber-shell area (not including the area corresponding to the void space in the cross section). Thermal analysis was performed using a thermogravimetric analyzer (PerkinElmer Pyris 1 TGA) and a differential scanning calorimeter (PerkinElmer Pyris Diamond DSC). An Axisymmetric Flow-Focusing Microfluidic Device** By Shoji Takeuchi, Piotr Garstecki, Douglas B. Weibel, and George M. Whitesides* This paper describes a microfluidic axisymmetric flowfocusing device (AFFD) fabricated in poly(dimethylsiloxane) (PDMS) that produces polymer-coated droplets with size distributions significantly more narrow than those generated using conventional microencapsulation methods.[1±3] The AFFD confines droplets in the central axis of a microfluidic channel; this confinement protects droplets from shear, or from damage re-COMMUNICATIONS Adv. Mater.