Analysis of Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

Research Area: A.1-Electron Microscopy Year: 2010
Type of Publication: Article
Publication: Journal of Bacteriology
Authors:
  • Fernando Amat
  • Luis R. Comolli
  • John F. Nomellini
  • Farshid Moussavi
  • Kenneth H. Downing
  • John Smit
  • Mark Horowitz
Abstract:
The surface layers (S-layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S-layer on intact growing cells of the gram negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, which increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between linear protein sequence and the structure at 2 nm resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative stain tomography proceeds from the N-terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C-terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.