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

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, and these 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 the linear protein sequence and the structure at the 2-nm level of 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.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that selforganizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7,14,43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the dif...

Global and Targeted Lipid Analysis of Gemmata obscuriglobus Reveals the Presence of Lipopolysaccharide, a Signature of the Classical Gram-Negative Outer Membrane

“…The proteinaceous barrier was previously suggested to substitute for other classical Gram-negative cell features, such as cell surface protection more typically provided by O antigen (20), a rigid structural framework in place of peptidoglycan (13), and possibly as an attachment platform for degradative enzymes, as in a related species, Rhodopirellula baltica (21). The proteinaceous exterior of G. obscuriglobus has not been shown to constitute a surface layer (S-layer), typically composed of only one or two proteins in a lattice configuration, like that found in some other bacteria, including archaea and the planctomycete "Candidatus Kuenenia stuttgartiensis" (13,22,23). The original cell wall analysis of G. obscuriglobus by Stackebrandt et al (18) found the cell wall composition to include many amino acids, and the ability of the G. obscuriglobus cell to maintain its integrity was demonstrated by the finding that cell sacculi retained their native cell morphology after 10% SDS treatment (18), thus exhibiting resistance to SDS that is more like that of cells that have a peptidoglycan-containing murein sacculus (21).…”

Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

“…The amino acid sequence specified for each segment is indicated in the supplemental material. The resulting plasmids were introduced by electroporation into JS1023, a C. crescentus NA1000 derivative (25).…”

S-layer Structure in Bacteria and Archaea

“…Negative staining, thinsection EM Smit et al (1992) Cryo-electron tomography (Cryo-ET) and statistical image processing Amat et al (2010) Arbing) appear to have the self-assembling or "crystallization domain" located in the Cterminal part of the protein, preceded by the cell-wall-anchoring domain(s), mostly SLH domains, at the N-terminus. The SLH domains are present in the majority of bacterial SLPs, though there are exceptions like SbsC in G. stearothermophilus (Pavkov et al 2008).…”