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Title: Unraveling the Structure-Function Relationships of Microbial Systems by High-Resolution in vitro Atomic Force Microscopy

Abstract

The elucidation of microbial surface architecture is critical to determining mechanisms of pathogenesis, immune response, physicochemical properties and environmental resistance. We have utilized in vitro AFM for studies of structure, assembly, function and environmental dynamics of several microbial systems including bacteria and bacterial spores. We have demonstrated, using various species of bacterial spores strikingly different species-dependent crystalline structures of the spore coat appear to be a consequence of crystallization mechanisms that regulate the assembly of the spore coat. Furthermore, we revealed molecular-scale transformations of the spore coat and cell outgrowth during the germination process. We will present data on the direct visualization of stress-induced environmental response of metal-resistant Arthrobacter oxydans bacteria to Cr (VI) exposure. These studies demonstrate that in vitro AFM can probe microbial surface architecture, environmental dynamics and the life cycle of pathogens at near-molecular resolution under physiological conditions.

Authors:
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
891055
Report Number(s):
UCRL-CONF-220631
TRN: US200621%%75
DOE Contract Number:
W-7405-ENG-48
Resource Type:
Conference
Resource Relation:
Conference: Presented at: Seeing at NanoScale IV, Philadelphia, PA, United States, Jul 17 - Jul 20, 2006
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ARCHITECTURE; ATOMIC FORCE MICROSCOPY; BACTERIA; BACTERIAL SPORES; CRYSTALLIZATION; GERMINATION; IN VITRO; LIFE CYCLE; PATHOGENESIS; PATHOGENS; PROBES; RESOLUTION; SPORES; TRANSFORMATIONS

Citation Formats

Malkin, A. Unraveling the Structure-Function Relationships of Microbial Systems by High-Resolution in vitro Atomic Force Microscopy. United States: N. p., 2006. Web.
Malkin, A. Unraveling the Structure-Function Relationships of Microbial Systems by High-Resolution in vitro Atomic Force Microscopy. United States.
Malkin, A. Thu . "Unraveling the Structure-Function Relationships of Microbial Systems by High-Resolution in vitro Atomic Force Microscopy". United States. doi:. https://www.osti.gov/servlets/purl/891055.
@article{osti_891055,
title = {Unraveling the Structure-Function Relationships of Microbial Systems by High-Resolution in vitro Atomic Force Microscopy},
author = {Malkin, A},
abstractNote = {The elucidation of microbial surface architecture is critical to determining mechanisms of pathogenesis, immune response, physicochemical properties and environmental resistance. We have utilized in vitro AFM for studies of structure, assembly, function and environmental dynamics of several microbial systems including bacteria and bacterial spores. We have demonstrated, using various species of bacterial spores strikingly different species-dependent crystalline structures of the spore coat appear to be a consequence of crystallization mechanisms that regulate the assembly of the spore coat. Furthermore, we revealed molecular-scale transformations of the spore coat and cell outgrowth during the germination process. We will present data on the direct visualization of stress-induced environmental response of metal-resistant Arthrobacter oxydans bacteria to Cr (VI) exposure. These studies demonstrate that in vitro AFM can probe microbial surface architecture, environmental dynamics and the life cycle of pathogens at near-molecular resolution under physiological conditions.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Thu Apr 06 00:00:00 EDT 2006},
month = {Thu Apr 06 00:00:00 EDT 2006}
}

Conference:
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  • Progress in structural biology very much depends upon the development of new high-resolution techniques and tools. Despite decades of study of viruses, bacteria and bacterial spores and their pressing importance in human medicine and biodefense, many of their structural properties are poorly understood. Thus, characterization and understanding of the architecture of protein surface and internal structures of pathogens is critical to elucidating mechanisms of disease, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. Furthermore, even though complete genome sequences are available for various pathogens, the structure-function relationships are not understood. Because of their lack ofmore » symmetry and heterogeneity, large human pathogens are often refractory to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). An alternative high-resolution method to examine native structure of pathogens is atomic force microscopy (AFM), which allows direct visualization of macromolecular assemblies at near-molecular resolution. The capability to image single pathogen surfaces at nanometer scale in vitro would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, specific cellular processes, environmental dynamics and biotransformation.« less
  • The elucidation of microbial surface architecture and function is critical to determining mechanisms of pathogenesis, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. We have utilized high-resolution in vitro AFM for studies of structure, assembly, function and environmental dynamics of several microbial systems including bacteria and bacterial spores. Lateral resolutions of {approx}2.0 nm were achieved on pathogens, in vitro. We have demonstrated, using various species of Bacillus and Clostridium bacterial spores, that in vitro AFM can address spatially explicit spore coat protein interactions, structural dynamics in response to environmental changes, and the life cycle ofmore » pathogens at near-molecular resolution under physiological conditions. We found that strikingly different species-dependent crystalline structures of the spore coat appear to be a consequence of nucleation and crystallization mechanisms that regulate the assembly of the outer spore coat, and we proposed a unifying mechanism for outer spore coat self-assembly. Furthermore, we revealed molecular-scale transformations of the spore coat during the germination process, which include profound, previously unrecognized changes of the spore coat. We will present data on the direct visualization of stress-induced environmental response of metal-resistant Arthrobacter oxydans bacteria to Cr (VI) exposure, resulting in the formation of a supramolecular crystalline hexagonal structure on the cell surface. At higher Cr (VI) concentrations the formation of microbial extracellular polymers, which cover microbial colony was observed. High-resolution visualization of stress-induced structures on bacterial surfaces builds a foundation for real time in vitro molecular scale studies of structural dynamics of metal-resistant bacteria in response to environmental stimuli. In the case of the bacterium Chlamedia trachomatis, we were able to identify surface exposed proteins versus proteins embedded in the outer membrane. These studies establish in vitro AFM as a powerful new tool capable of revealing pathogen architecture, structural dynamics and variability at nanometer-to-micrometer scales.« less
  • High-speed atomic force microscopy has attracted much attention due to its unique capability of visualizing nanoscale dynamic processes at a solid/liquid interface. However, its usability and resolution have yet to be improved. As one of the solutions for this issue, here we present a design of a high-speed Z-tip scanner with screw holding mechanism. We perform detailed comparison between designs with different actuator size and screw arrangement by finite element analysis. Based on the design giving the best performance, we have developed a Z tip scanner and measured its performance. The measured frequency response of the scanner shows a flatmore » response up to ∼10 kHz. This high frequency response allows us to achieve wideband tip-sample distance regulation. We demonstrate the applicability of the scanner to high-speed atomic-resolution imaging by visualizing atomic-scale calcite crystal dissolution process in water at 2 s/frame.« less
  • The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging. P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structuralmore » analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.« less