Assembly of Xylanases into Designer Cellulosomes Promotes Efficient Hydrolysis of the Xylan Component of a Natural Recalcitrant Cellulosic Substrate

In nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose component of the plant cell wall is embedded in a colloidal blend of hemicelluloses, a major component of which is xylan. In order to enhance enzymatic degradation of the xylan component of a natural complex substrate (wheat straw) and to study the synergistic action among different xylanases, we have employed a variation of the designer cellulosome approach by fabricating a tetravalent complex that includes the three endoxylanases of Thermobifida fusca (Xyn10A, Xyn10B, and Xyn11A) and an Xyl43A β-xylosidase from the same bacterium. Here, we describe the conversion of Xyn10A and Xyl43A to the cellulosomal mode. The incorporation of the Xyl43A enzyme together with the three endoxylanases into a common designer cellulosome served to enhance the level of reducing sugars produced during wheat straw degradation. The enhanced synergistic action of the four xylanases reflected their immediate juxtaposition in the complex, and these tetravalent xylanolytic designer cellulosomes succeeded in degrading significant (~25%) levels of the total xylan component of the wheat straw substrate. The results suggest that the incorporation of xylanases into cellulosome complexes is advantageous for efficient decomposition of recalcitrant cellulosic substrates—a distinction previously reserved for cellulose-degrading enzymes

Lysozyme activity of the Ruminococcus champanellensis cellulosome

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135571/1/emi13501.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135571/2/emi13501_am.pd

Sequence-to-Sequence Alignment

This thesis studies the problem of spatio-temporal alignment of video sequences, i.e., establishing correspondences in time and in space between two di erent video sequences of the same dynamic scene. It shows that temporal variations between image frames such as moving objects, changes in scene illumination, or camera ego-motion, are powerful cues for alignment. Such temporal variations cannot be exploited by standard image-to-image alignment techniques, as they are not captured by a single image, but only by a sequence of images. We show that by folding these new temporal cues and known spatial cues into a single alignment framework, situations which are inherently ambiguous for traditional image-to-image alignment methods are often uniquely resolved by sequence-to-sequence alignment. This gives rise to a wide range of new video applications. These are discussed in this thesis. The thesis investigates the cases where thesequences are recorded byuncalibrated video cameras with xed internal and relative external parameters. However, the notion of sequence-to-sequence alignment/matching is more general, and is not restricted to those cases alone.

Deformation of Filamentous <i>Escherichia coli</i> Cells in a Microfluidic Device: A New Technique to Study Cell Mechanics

<div><p>The mechanical properties of bacterial cells are determined by their stress-bearing elements. The size of typical bacterial cells, and the fact that different time and length scales govern their behavior, necessitate special experimental techniques in order to probe their mechanical properties under various spatiotemporal conditions. Here, we present such an experimental technique to study cell mechanics using hydrodynamic forces in a microfluidic device. We demonstrate the application of this technique by calculating the flexural rigidity of non-growing <i>Escherichia coli</i> cells. In addition, we compare the deformation of filamentous cells under growing and non-growing conditions during the deformation process. We show that, at low forces, the force needed to deform growing cells to the same extent as non-growing cells is approximately two times smaller. Following previous works, we interpret these results as the outcome of the difference between the elastic response of non-growing cells and the plastic-elastic response of growing cells. Finally, we observe some heterogeneity in the response of individual cells to the applied force. We suggest that this results from the individuality of different bacterial cells.</p></div

Analysis of the deformation of non-growing cells.

<p>The difference of the angle between the tip of the cell () and its basal end () for growing cells. The value of was derived from a global fit to a straight line of for , , , , and cells at infusion rates of , , , , and , respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083775#pone.0083775.s005" target="_blank">figure S5</a> ). Gray line - fit to an exponential function.</p

Sketch of the experimental setup.

<p>Cells, e.g. cell (i) and (ii), grew in a microfluidic device consisting of dead-end growth channels that were connected at their open end to a large main channel. If cell division is blocked, the cells grow as filaments that penetrate into the main channel. A fluid flow with velocity and a profile similar to the one depicted created a hydrodynamic force that deformed the cells, as is shown for cell (ii). The magnitude of the force was controlled by the infusion rate. For analysis, the arc length () was measured from the point of connection between the growth channels and the main channel () to the tip of the cell (). To characterize the shape of the cell, the angle profile () was calculated (see Materials and Methods).</p