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De Novo synthesis of the cell wall in E. coli: Reversion of L-Forms.
G. H. Billings1, K. C. Huang2; 1Department of Physics, Stanford University, Stanford, CA, 2Department of Bioengineering, Stanford University, Stanford, CA
Bacterial morphology is crucial to a wide variety of cell functions, including motility, adhesion, pathogenesis. The shape of nearly all bacterial cells is determined by a rigid cell wall made of peptidoglycan. A rod-shaped cell such as E. coli thus faces the challenge of coordinating the nanoscale proteins responsible for peptidoglycan synthesis to construct a micron-scale sacculus. What are the principles that allow cell wall synthesis proteins to establish order over a range of length scales spanning nearly three orders of magnitude? We approached this question by examining the re-growth of the sacculus in cell wall-deficient 'L-forms' of E. coli, in which cell wall synthesis has been transiently disrupted by beta-lactam antibiotics. An L-form undergoing reversion begins in a spherical shape without an intact cell wall. When cell wall synthesis inhibiting antibiotics are removed, the cell generates new rod-shaped protrusions, which eventually undergo septation and adopt the normal rod morphology of E. coli. The
reversion of L-forms thus provides an opportunity to study morphogenesis in bacteria lacking an intact cell wall, and to study the activity of cell wall synthesis enzymes independently of their interaction with the cell wall. We used automated image analysis to quantify the morphological trajectories of reverting L-forms as they transition from rod to sphere, and to track the activity of cell wall synthesis enzymes. In particular, we examined the localization of bacterial cytoskeletal elements MreB and FtsZ, both of which are involved in cell wall growth and morphogenesis, and correlated their localization patterns with changes in cell shape. We also probed the effect of different mutants of MreB on the dynamics of the reversion process. We found that MreB determines the size of early rod-shaped protrusions in reverting L-forms, demonstrating that MreB regulates cell shape even in the absence of an intact cell wall.
178
MinCD cell division proteins form alternating co-polymeric filaments.
D. Ghosal1, L. A. Amos1, J. Löwe1; 1Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Most bacteria form a dynamic ring-like structure at the division site, containing the tubulin-like protein FtsZ, which constricts to produce two daughter cells. The Min proteins play an important role in the precise positioning of Z ring at the division site by oscillating between the poles in most organisms.
In E. coli, the three proteins MinC, MinD and MinE, encode the Min system. MinC and MinD are widely conserved in bacteria and they act together to inhibit Z ring assembly. MinD binds to the membrane in dimeric form in the presence of ATP through an amphipathic helix and also binds to MinC. MinC directly interacts with FtsZ and destabilises Z ring assembly. MinE stimulates the ATP hydrolysis of MinD and hence dissociates the MinCD complex from the membrane. So far it has been thought that a reaction/diffusion pattern-forming reaction based on MinD and MinE alone causes the oscillation of all three proteins.
Using light scattering and electron microscopy (EM) we show in vitro that MinC and MinD form nucleotide-dependent, alternating co-polymeric filaments, even in the absence of membrane. To better understand the interaction between MinC and MinD in the filament, we have solved the crystal structure of the MinC-MinD complex at 2.7 Å resolution. A hybrid model of the MinCD filaments, built using the MinCD and MinD dimer crystal structures correlates well with the appearance of MinCD filaments as observed in EM. Our structural analysis also identifies residues important for the MinCD interaction and helps to explain how MinE competes with MinC for the overlapping binding site on MinD.
We propose that the MinCD filament belongs to a new class of cytomotive, nucleotide-regulated filaments, adding to actin and tubulin-like systems present in many bacteria.
179
How to segregate DNA without dynamic instability: Bundling of the bacterial actin-like filament AlfA is regulated by an accessory factor, AlfB.
J. K. Polka1, R. D. Mullins1; 1UCSF, San Francisco, CA
AlfA is a filament-forming actin-like protein in Bacillus subtilis that functions to actively partition the large, low copy number plasmid by which it is encoded. Previous in vivo and in vitro observations of filament dynamics have revealed a set of kinetic and structural properties (namely constitutive bundling and lack of dynamic instability) that are inconsistent with previously established models for actin-like plasmid segregating proteins such as ParM. To
understand the mechanism of AlfA-driven plasmid segregation, we imaged AlfA and its downstream DNA-binding protein, AlfB, interacting with plasmids in vivo and in vitro. Our live cell microscopy reveals that plasmids can move along existing AlfA structures or track the ends of growing ones, consistent with the idea that the AlfA polymer seen in vivo is actually a bundle of multiple filaments. Furthermore, these polymers can form between plasmids to push them apart, prompting us to ask how plasmids alter filament dynamics to generate this specific assembly. To address this question, we purified AlfB and found that it dramatically alters the kinetics and structure of AlfA. AlfB binds to AlfA monomers and polymers, not only increasing the critical concentration of assembly, but also preventing the otherwise very robust bundling of AlfA. The 100bp centromeric DNA region to which AlfB binds, however, rescues bundling and promotes polymerization. These observations lead us to a model of AlfA-driven plasmid segregation wherein bundles of AlfA form specifically in association with AlfB-DNA complexes. We propose that the intrinsic bundling property of the polymer, normally inhibited by a high concentration of free AlfB in the cytoplasm, functions as a capture mechanism to specifically join DNA-bound filaments to one another. Polymerization in opposite directions, driven by antiparallel bundling, would cause plasmids to be segregated from one another, ensuring their maintenance through cell division.
180
Spatial regulation of protein distribution in bacterial cells.
S. Schlimpert1,2, E. A. Klein3, A. Briegel4, V. Hughes5, J. Kahnt6, K. Bolte2, U. G. Maier7, Y. V. Brun5, G. J. Jensen4,8, Z. Gitai3, M. Thanbichler1,2; 1Max Planck Institute for Terrestrial
Microbiology, Marburg, Germany, 2Faculty of Biology, Philipps-Universität, Marburg, Germany, 3
Department of Molecular Biology, Princeton University, Princeton, NY, 4Division of Biology, California Institute of Technology, Pasadena, CA, 5Department of Biology, Indiana University, Bloomington, IN, 6Department of Ecophysiology, Max Planck Institute for Terrestrial
Microbiology, Marburg, Germany, 7Department of Biology, Philipps-Universität, Marburg, Germany, 8Howard Hughes Medical Institute, Pasadena, CA
The formation of specialized cellular architectures often requires the establishment and maintenance of subcellular domains with a distinct lipid and protein composition. We report the identification of a novel mechanism that determines the distribution of proteins within cells of the stalked model bacterium Caulobacter crescentus. It depends on the establishment of a macromolecular protein complex forming at the junction between the cell body and the stalk appendage. Mutants with defects in the assembly or integrity of this structure show aberrant protein distribution patterns, which in turn lead to a significant decrease in cellular fitness. Collectively, our results demonstrate that eukaryotes and prokaryotes use similar molecular principles for the regulation of subcellular protein localization in order to ensure the proper function of cells.
181
Time-resolved nanometer scale AFM imaging of antimicrobial peptide activity on live Escherichia coli cells.
A. Slade1, J. H. Kindt1, S. C. Minne1; 1Bruker Nano Inc., Santa Barbara, CA
Understanding drug-membrane interactions is crucial to drug research and development. Bacterial membranes have a much more complex structure than mammalian cell membranes. As such, knowledge of bacterial membrane composition and organization, as well as characterization of the molecular-level responses to drug interactions, is critical to the development and assessment of effective drug formulations. Cellular drug responses involve
highly dynamic processes. However, the ability to image live cells with nanometer resolution on timescales relevant to dynamic cellular events has proven challenging. The ability of atomic force microscopy (AFM) to obtain three-dimensional topography images of biological molecules with nanometer resolution and under near-physiological conditions remains unmatched by other imaging techniques. However, with traditional AFM systems, the typically longer image acquisition times required to obtain a single high-resolution image (~minutes) has limited the ability to investigate dynamic biological processes. While recent years have shown significant progress in the development of high-speed atomic force microscopy (HS-AFM), the nature of the instrumentation that has been developed has several drawbacks in specimen size, requiring small scan sizes and flat sample surfaces. As such, the majority of biologically-related HS-AFM studies have concentrated on imaging single biomolecules with little focus on using HS-AFM to examine cellular processes. With the rapidly growing antibiotics crisis, antimicrobial peptides (AmP) are increasingly being investigated as therapeutic alternatives. Key to their success is an understanding of the mechanisms by which AmPs interact with the cell membrane and facilitate cellular death. Using HS-AFM, we have obtained the first high-resolution time sequence images of the native structure of a bacterial outer membrane, obtained directly on the surface of live Escherichia coli cells. The increased time resolution of HS-AFM allowed us to observe dynamic changes in the nanoscale structure of the outer membrane in direct response to the AmP CM15, at timescales relevant to the mechanism of AmP-induced cell death. The results of these HS- AFM studies have provided the first opportunity to resolve the dynamics of AmP-mediated cell death in a native cell membrane environment in real-time and with nanoscale resolution.
182
Type 6 secretion dynamics within and between bacterial cells.