The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy

Abstract

The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA4 gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

“…It is estimated that 60 000 RsaA monomers are interlinked to form the S-layer. The correct crystallization of the structure into its native hexagonal pattern requires calcium ions (Gilchrist et al, 1992;Smit et al, 1992;Walker et al, 1992Walker et al, , 1994Bingle et al, 1997). Secretion of the protein relies on a stable C-terminal secretion signal (Bingle et al, 1997) and a three-component bacterial ABC-transporter system, two components of which have been cloned and sequenced (P. Awram and J.…”

Factors controlling in vitro recrystallization of the Caulobacter crescentus paracrystalline S-layer

“…The structure is a two-dimensional highly ordered array that is attached to the outer membrane, has no portion that penetrates the outer membrane, and completely covers the bacterium. It is composed of a single protein species with a size of 98 kDa (11) which self assembles into a hexagonally arranged lattice; the prominent morphological unit contains six monomers and is spaced at 22-nm intervals (28). We study the S-layer gene (rsaA) and protein (RsaA) of C. crescentus CB15 in an effort to define key features of this structure, that is, the factors that control secretion, surface attachment, and self-assembly of the protein into a two-dimensional crystalline structure.…”

“…Moreover, it is, in principle, possible to get long-range good order from RsaA only. When colonies derived from mutants that fail to attach the S-layer (due to the loss of a specific species of lipopolysaccharide [LPS] termed smooth LPS [SLPS] [31]) are examined, extensive sheets of S-layer which form as a mirror-image double layer of S-layer and show a high degree of order are found (28,31).In vitro recrystallization has been accomplished with a variety of other bacterial S-layer systems (15,(23)(24)(25); often S-layer proteins derived from gram-positive bacteria will reassemble after simple removal of the dissociating agent (usually a chaotrope), with no requirements for ions or a particular surface. In…”

“…Moreover, it is, in principle, possible to get long-range good order from RsaA only. When colonies derived from mutants that fail to attach the S-layer (due to the loss of a specific species of lipopolysaccharide [LPS] termed smooth LPS [SLPS] [31]) are examined, extensive sheets of S-layer which form as a mirror-image double layer of S-layer and show a high degree of order are found (28,31).…”

Alphabet SOUP: A framework for approximate energy minimization

“…We experiment with this approach over the task of reconstructing the outermost surface layer (S-layer) of a bacteria from images obtained by 3D-tomography from cryoelectron microscopes. The S-layer often exhibits geometrical lattice-like 2D structure [23,22], which provides insight into how the bacteria interacts with its environment. These structures are not easily visible in the raw 2D images, but much more easily discerned in a 3D surface reconstruction.…”