Small-Angle X-Ray Scattering for Imaging of Surface Layers on Intact Bacteria in the Native Environment

Abstract

Crystalline cell surface layers (S-layers) represent a natural two-dimensional (2D) protein self-assembly system with nanometerscale periodicity that decorate many prokaryotic cells. Here, we analyze the S-layer on intact bacterial cells of the Gram-positive organism Geobacillus stearothermophilus ATCC 12980 and the Gram-negative organism Aquaspirillum serpens MW5 by smallangle X-ray scattering (SAXS) and relate it to the structure obtained by transmission electron microscopy (TEM) after platinum/ carbon shadowing. By measuring the scattering pattern of X rays obtained from a suspension of bacterial cells, integral information on structural elements such as the thickness and lattice parameters of the S-layers on intact, hydrated cells can be obtained nondestructively. In contrast, TEM of whole mounts is used to analyze the S-layer lattice type and parameters as well as the physical structure in a nonaqueous environment and local information on the structure is delivered. Application of SAXS to S-layer research on intact bacteria is a challenging task, as the scattering volume of the generally thin (3-to 30-nm) bacterial S-layers is low in comparison to the scattering volume of the bacterium itself. For enhancement of the scattering contrast of the S-layer in SAXS measurement, either silicification (treatment with tetraethyl orthosilicate) is used, or the difference between SAXS signals from an S-layer-deficient mutant and the corresponding S-layer-carrying bacterium is used for determination of the scattering signal. The good agreement of the SAXS and TEM data shows that S-layers on the bacterial cell surface are remarkably stable. Surface layers (S-layers) are a distinct cell surface decoration comprised of regularly arrayed, two-dimensional (2D) protein or glycoprotein crystals present on many prokaryotic organisms from almost all known phylogenetic branches (1-4). S-layers form upon self-assembly and exhibit nanometer-scale periodicity with oblique (p1, p2), square (p4), or hexagonal (p3, p6) lattice symmetry and species-specific lattice constant values in the range of 10 to 25 nm. The S-layer self-assembly system holds great promise as a bottom-up approach for organizing matter at the nanometer scale as required in the field of nanobiotechnology (5).Because of their cell surface location, ultrastructural studies of S-layer lattices have been frequently performed by high-resolution transmission electron microscopic (TEM) methods such as ultrathin sectioning or heavy-metal shadow casting of freezeetched or freeze-dried whole cells (6) as well as image analysis (7) or cryo-sectioning of frozen-hydrated cells (8). These electron microscopic approaches, except the latter, suffer from the drawback of the requirement for water-free specimens, implying a requirement for specific sample treatment prior to analysis. The standard procedures of chemical fixation, dehydration, and staining of cells for TEM analysis may affect cell morphology and ultrastructure and, thus, prevent the accurate determination of distinct S-l...

Characterizing the S‐layer structure and anti‐S‐layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X‐ray scattering

“…For SAXS data evaluation, the value for wild-type cells was subtracted from the value for S-layer-deficient cells. After division of the signal by the form factor for infinite plates, the intensity modulations were interpreted as the structure factor to determine the size and arrangement of the two glycoproteins TfsA-GP and TfsB-GP (Sekot et al ., 2013).…”

“…Small-angle X-ray scattering (SAXS) offers another option for determining the structure of biological macromolecules in different environments under conditions close to those in physiological settings (Svergun and Koch, 2003). Its application to S-layer research on intact bacteria was demonstrated recently (Sekot et al ., 2013).…”

Tannerella forsythiastrains display different cell-surface nonulosonic acids: biosynthetic pathway characterization and first insight into biological implications

“…After the T. forsythia S-layer had been found to delay the host immune response, at least at the early stage of infection, (Sekot et al 2011), a detailed investigation of the bacterium’s cell surface architecture (Sekot et al 2013) and glycosylation ensued (Posch et al 2011). As previously mentioned, the S-layer of the ATCC 43037 type strain was shown to be modified with an O -linked oligosaccharide containing several rare sugar residues (Figure 1B), including a Pse derivative with an N -acetimidoyl (Am) group at C-5 and an N -glyceroyl (Gra) group at C-7 (Pse5Am7Gra; Figure 1A; (Sekot et al 2011)).…”

Comparative genome characterization of the periodontal pathogen Tannerella forsythia

“…Examples include welldescribed virulence factors such as the leucine-rich-repeat protein BspA [2,3] and the protease PrtH/Fdf [4]. The T. forsythia cell surface (S-) layer was described to consist of the alternating TfsA and TfsB glycoproteins that have their corresponding genes located next to each other in the genome [5][6][7] and align in a 2D lattice, which drastically impacts the host immune response [8][9][10]. In T. forsythia, the S-layer proteins as well as other cell surface proteins are modified with a complex O-glycan that can be dissected in a species-specific portion and a core saccharide that is proposed to be conserved in the Bacteroidetes phylum of bacteria [6,10,11].…”