Structural and chemical characterization of the S layer of a Pseudomonas-like bacterium

Abstract

Sections and freeze-fractured preparations showed an S layer on the surface ofPseudomonas-like strain EU2. Polyacrylamide gel electrophoresis of cell envelopes extracted with 1% sodium dodecyl sulfate (SDS) at room temperature showed three proteins (45K, 55K, and 110K). The 55K protein was identified as the S-layer protein. Incubation in 1.5 M guanidine hydrochloride removed the S layer from cell envelopes and dissociated the structure into subunits. The soluble 55K protein reassembled into planar sheets upon removal of the guanidine hydrochloride by dialysis. Electron microscopy and image processing indicated that these sheets had p4 symmetry in projection with a lattice constant of 13.2 ± 0.1 nm (corresponding to 9.3 nm between adjacent fourfold axes). In some instances these reassemblies appeared to form small three-dimensional crystals which gave particularly clear views of the structure in projection because of the superimposition of information from a number of layers. A model is proposed with molecules having rounded lobes connected by a narrower linker region and joining at the lobes to form the fourfold axes of the array. The pattern superficially resembles those of other bacterial S layers, such as those of Aeromonas salmonicida, Aeromonas hydrophila, and Azotobacter vinelandii. Extraction of cell envelopes with 1% SDS at 50°C released the 110K protein from the envelopes and removed an amorphous backing layer from the S layer. The 45K protein displayed heat-modifiable migration in SDS-polyacrylamide gel electrophoresis and was insoluble in SDS at 50°C or in high concentrations of guanidine hydrochloride, suggesting that it was associated with the peptidoglycan.S layers form regular arrays on the external surfaces of many bacteria. These layers often consist of the highestmolecular-weight protein in cells possessing such layers, and that protein is present in higher amounts than any other cellular protein. They may also perform important protective functions in the bacteria, possibly related to their forming a barrier to molecules (enzymes) and predators (35). S layers have also been suggested to form specific cell-cell connections involving alignment of the pores to form continuous channels between cells (5). They are found in a wide variety of eubacteria and archaebacteria (35,(45)(46)(47) and are arranged in p2, p4, or p6 symmetry with lattice spacings ranging from 2.8 to 35 nm. The protein subunits range in molecular weight from 13,000 to 255,000 (45, 46) and assemble in multimeric units, usually held together by noncovalent bonds including hydrogen bonds, hydrophobic bonds, and ionic bonds (45,46). Determination of the three-dimensional structure of S layers is becoming increasingly common (30) and reveals a high degree of variability of structure on the outer surfaces, whereas the inner surfaces of S layers tend to be more conserved (4), at least to the ca. 2-nm resolution of these studies.Although S layers have been recognized in a great number of species from most phylogenetic lines, very few S ...

Low dose aerosol infection of mice with virulent type A Francisella tularensis induces severe thymus atrophy and CD4+CD8+ thymocyte depletion

“…Sections of 4 μm thickness were stained with hematoxylin and eosin (HE), and examined by light microscopy. Preparation for transmission electron microcopy (TEM) essentially followed the method of Austin et al [35]. Tissues were fixed in 0.2 M cacodylate buffer pH 7.4 containing 2.5% (v/v) glutaraldehyde, post-fixed in 0.2 M cacodylate buffer pH 7.4 containing 1% OsO 4 , and dehydrated through a graded series of ethanol (15 min each in 50, 70, and 90% ethanol, followed by three 20 min incubations in 100% ethanol).…”

Attachment of a Pseudomonas-like bacterium and Bacillus coagulans to solid surfaces and adsorption of their S-layer proteins

“…The S-layer of EU2 is a 55 kDa protein, which assembles into a tetragonal array with a lattice constant of 13.2 & 0.1 nm and which can form three-dimensional crystals (Austin et al, 1990). Its pattern resembles those of Aeromonas salmonicida, Aeromonas hydrophila and Azotobacter vinelandii (Austin et al, 1990). B. coagulans, in contrast, has a 100 kDa monomer S-layer protein which, when assembled, exhibits oblique symmetry (Pum et al, 1989).…”

“…(6.89 MPa). Suspensions were centrifuged at 20000 g for 20 min, and the pellet was treated as described by Austin et al (1990), except that final dialysis was against 100 m~-HEPES containing 7.5 m~-CaCl, overnight at 4 "C with stirring, to remove the guanidine hydrochloride used to extract the S-layer. The precipitate, which formed after dialysis, represented the self-assembled 55 kDa S-layer protein.…”

“…The chemical structures of EU2 and B. coagulans S-layers are quite different. The S-layer of EU2 is a 55 kDa protein, which assembles into a tetragonal array with a lattice constant of 13.2 & 0.1 nm and which can form three-dimensional crystals (Austin et al, 1990). Its pattern resembles those of Aeromonas salmonicida, Aeromonas hydrophila and Azotobacter vinelandii (Austin et al, 1990).…”

“…Thus, the surfaces had the same basic chemistry, yet differed in hydrophobicity, which could be expected to influence protein adsorption (Norde, 1986) and bacterial adhesion (Rosenberg & Kjelleberg, 1986). The organisms were a Gram-negative Pseudomonas-like strain, designated EU2 (Austin et al, 1990) and the Gram-positive thermophile Bacillus coagulans (pum et al, 1989). The chemical structures of EU2 and B. coagulans S-layers are quite different.…”