Ultrastructure, inferred porosity, and gram-staining character of Methanospirillum hungatei filament termini describe a unique cell permeability for this archaeobacterium

Abstract

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.

Transmission electron microscopy, scanning tunneling microscopy, and atomic force microscopy of the cell envelope layers of the archaeobacterium Methanospirillum hungatei GP1

“…11 and 12). STM has confirmed the outermost plug layer to be of the particulate variety (4), since all intact end plugs viewed by this technique had particulate surfaces. Furthermore, STM images produced better height resolution (Fig.…”

“…The height of the end plugs varied according to the number of component layers. Although intact end plugs contain multiple lamellae, these layers slip and separate from one another during their isolation (4) so that this preparation is a dispersion of unequal plug composites. It is possible that the amorphous material is the "glue" which holds the lamellae together, and its dissolution during end plug purification may be responsible for the separation.…”

“…The end plug is a multilaminar structure which contains two different types of disc-shape proteinaceous assemblies (4). Both of these individual plug layers possess 18-nm repeats with different p6 packing arrangements: a particulate layer consisting of large, roughly circular 14-nm subunits, and a holey layer consisting of roughly circular pores (ca.…”

Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1

“…Its ability to withstand various dissolution conditions (5) has been attributed to the presence of strategic disulfide bonds (20,21). The function of the sheath as a physical barrier is implied by this resilience and also by the ability of the filaments to exclude the crystal violet of the Gram stain (4). The sheath is also a rigid structure (cylinder), presumably because of covalent bonding in combination with weaker bonds (e.g., ionic bonding and hydrophobicity).…”

“…The sheath is structurally unique, possessing a 2.8-nm paracrystalline repeat overlying a generally amorphous inner region (5,24). On the basis of the presence of this 2.8-nm repeat, the sheath is thought to function as a sieve and to exclude molecules larger than acetate (24) or crystal violet (4). For the substrate, this would allow the transfer of the basic nutrients of the bacterium (hydrogen, carbon dioxide, and acetate) into cells and the transfer of its primary metabolic product (methane) out of cells.…”