By light microscopy, Methanospirillum hungatei GP1 stains gram positive at the terminal ends of each multicellular filament and gram negative at all regions in between. This phenomenon was studied further by electron microscopy and energy-dispersive X-ray spectroscopy of Gram-stained cells, using a platinum compound to replace Gram's iodine (J. A. Davies, G. K. Anderson, T. J. Beveridge, and H. C. Clark, J. Bacteriol. 156:837-845, 1983). Crystal violet-platinum precipitates could be found only in the terminal cells of each filament, which suggested that the multilamellar plugs at the filament ends were involved with stain penetration. When sheaths were isolated by sodium dodecyl sulfate-dithiothreitol treatment, the end plugs could be ejected and their layers could be separated from one another by 0.1 M NaOH treatment. Each plug consisted of at least three individual layers; two were particulate and possessed 14.0-nm particles hexagonally arranged on their surfaces with a spacing of a = b = 18.0 nm, whereas the other was a netting of 12.5-nm holes with spacings and symmetry identical to those of the particulate layers. Optical diffraction and computer image reconstruction were used to clarify the structures of each layer in an intact plug and to provide a high-resolution image of their interdigitated structures. The holes through this composite were three to six times larger than those through the sheath. Accordingly, we propose that the terminal plugs of M. hungatei allow the access of larger solutes than does the sheath and that this is the reason why the end cells of each ifiament stain gram positive whereas more internal cells are gram negative. Intuitively, since the cell spacers which partition the cells from one another along the filament contain plugs identical in structure to terminal plugs, the diffusion of large solutes for these cells would be unidirectional along the filament-cell axis.
Negative staining revealed a tetragonal surface array (S layer) on all the members of a serogroup of Aeromonas hydrophila which possess high virulence for fish. The S layers were similar on all the strains examined, with unit cell dimensions of approximately 12 nm. A single representative strain, strain TF7, was selected for further analysis. Freeze-cleaved and etched aggressive and are considered to be opportunistic fish pathogens. The virulent strains all belonged to a single heat-stable serogroup and possessed unique cell surface-associated phenotypic properties (21). Strains of A. hydrophila associated with invasive diseases of humans also display these phenotypic characteristics (13). We examined the members of this serogroup with high virulence for fish by a number of electron-microscopic techniques and found that each strain possessed a tetragonal S layer. The surface-associated, regularly structured S layers have so far been described for only a small number of pathogenic bacteria including the related gram-negative fish pathogen A. salmonicida (12), for which these layers are recognized as an essential virulence factor. S layers are ideally situated to influence the outcome of a host-parasite encounter because of the cell surface location. They have been implicated as a permeability barrier (24,25) It is therefore necessary to define the nature of the surface of these virulent A. hydrophila strains at both the morphological and the biochemical levels as a contribution to understanding the biological functions and possible role of the surface in pathogenesis. In this report we describe the subunit arrangement in the paracrystalline surface array in a representative strain pathogenic for salmonid fish, A. hydrophila TF7. MATERIALS AND METHODSBacterial strains and growth conditions. The A. hydrophila strains examined in this study and their sources are listed in Table 1. Stock cultures were maintained at -70°C in 15%
Electron microscopy of the Azotobacter vinelandii tetragonal surface array, negatively stained with ammonium molybdate in the presence of 1 mM calcium chloride, showed an apparent repeat frequency of 12 to 13 nm. Image processing showed dominant tetrad units alternating with low-contrast cruciform structures formed at the junction of slender linkers extending from corner macromolecules of four adjoining dominant units. The actual unit cell showed p4 symmetry, and a = b = 18.4 nm. Distilled water extraction of the surface array released a multimeric form of the single 60,000-molecular-weight protein (S protein) which constitutes the surface layer. The molecular weight of the multimer was estimated at 255,000 by gel filtration, indicating a tetrameric structure of four identical subunits and suggesting that this multimer was the morphological subunit of the S layer. Tetrameric S protein exhibited low intrinsic stability once released from the outer membrane, dissociating into monomers when incubated in a variety of buffers including those which served as the base for defined media used to cultivate A. vinelandii. The tetramer could not be stabilized in these buffers at any temperature between 4 and 30°C, but the addition of 2 to 5 mM Ca2+ or Mg2+ completely prevented its dissociation into monomers. Circular dichroism measurements indicated that the secondary structure of the tetramer was dominated by aperiodic and 18-sheet conformations, and the addition of Ca2' did not produce any gross changes in this structure. Only the tetrameric form of S protein was able to reassemble in vitro in the presence of divalent cations onto the surface of cells stripped of their native S layer.
Measurements of the low temperature luminescent spectra of CaF2:Er3+ have revealed a vibronic sideband associated with the Er3+ electronic transition at 5512 Å. The phonon frequencies observed are shown to be related to the single phonon density of states of CaF2.
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