Ultrastructure of the cell envelope of the archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus

Abstract

The ultrastructures of the regular surface layers (S-layers) of the extremely thermophilic archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus were examined by freeze-etching, freeze-drying, and negative staining methods combined with optical and digital image enhancement. In both strains, a monolayer of macromolecules arranged in hexagonal arrays with center-to-center spacings of approximately 30 nm was the only component of the cell wall. The gross morphologies of the S-layer lattices of the two organisms were similar and showed the same handedness in the arrangement of the protomers of the morphological units. Striking differences were found in the anionic charge distributions on the surfaces of the two S-layer proteins as determined by labeling with polycationic ferritin. Analysis of the lattice orientation, together with the number and distribution of lattice faults on intact cells, provided a strong indication that the S-layers of both organisms have a shape-determining function.Thermoproteus tenax (29) and Thermoproteus neutrophilus (6) are members of a recently discovered, novel order of extremely thermophilic anaerobic archaebacteria. They are hydrogen-sulfur autotrophic and grow at temperatures of up to 95°C (25). T. tenax differs from T. neutrophilus in that it can also grow heterotrophically and has an acidic pH growth optimum rather than one close to neutral (6). Their light microscopical appearance is that of extended aseptate rods of about 0.4 ,um in diameter with a length of 1.0 to 80 p.m. T. tenax often shows true branching (29). The bacteria are nonmotile and show a gram-negative staining reaction due to the simple wall architecture without a murein or pseudomurein layer (11,12,29). The walls consist of the cytoplasmic membrane and a hexagonally packed surface layer (S-layer) (22,24).We have investigated the ultrastructure and surface charge of T. tenax and T. neutrophilus, firstly to obtain information about proteins (13) which are able to withstand extreme environmental conditions and, secondly, to elucidate a possible morphogenetic function of S-layers in organisms in which paracrystalline arrays are the only component of the cell wall.MATERIALS AND METHODS Bacteria. T. tenax (DSM 2078) was isolated from a solfataric mud hole in the Kraffla area, Iceland. T. neutrophilus (DSM 2338) was isolated from a hot spring in the Kerlingarfj6ll, Iceland. Both species were grown while stirred in Allen medium (1) supplemented with 0.5% sulfur and 0.02% yeast extract. The cultures were gassed with a mixture of 80% hydrogen and 20% carbon dioxide. T. tenax was grown at pH 5.5, and T. neutrophilus was grown at pH 6.8.Materials. Cationized ferritin was purchased from Sigma Chemical Co., Munich, Federal Republic of Germany. All other chemicals used were of reagent grade.Specimen preparation. For freeze-etching, the whole bacteria were centrifuged, and 1-pl samples were immediately frozen in Freon 22 (Polaron Equipment Ltd., Watford, England) kept close to its solidification temperature * Corresp...

Crystalline surface layers in procaryotes

“…Formation of the S-layers of Thermoproteus tenax and Thermoproteus neutrophilus (53) and of the sheathlike S-layer of Methanospirillum hungatei (12,69), Methanothrix concilji (57) and Methanothrix soehngenii (35), all of which are highly resistant to disintegrating agents (see above), can be considered atypical; their assembly must involve the selective breaking and formation of covalent, intersubunit bonds at well-defined areas. Lattice faults (pentameric units resembling wedge disclinations) identified in the hexagonal arrays at the cell poles of T. tenax could represent such specific subunit incorporation sites for elongation growth of the cylindrical part of the cell (53). FUNCTION Considering that procaryotes carrying S-layers are ubiquitous in the biosphere, the supramolecular concept of a "porous crystalline surface layer" composed of identical protein or glycoprotein subunits would have the potential to fulfill a broad spectrum of functions.…”

“…Such an "archaic" functional principle appears to be preserved in those archaebacteria which have S-layers as the only, and occasionally quite rigid, wall component (e.g., Thermoproteus spp. [53], the "square bacterium" [91], H. halobium [85,97], and H. salinarium [51]) and maintain a well defined rod, filamentous, or "square" morphology during cell growth and in those methanogens which possess a sheath (e.g., Methanospirillum hungatei [12,35], Methanothrix concilii [57], and Methanothrix soehngenii [35]). Further support for this notion comes from the fact that these primitive envelope structures are found in organisms which branched off very early in the phylogenetic tree (75a, 98).…”

“…Many S-layers require monovalent or bivalent cations for structural integrity (8,37). Common [35,53] and for sheathlike S-layers such as those in Methanothrix soehngenii [35], Methanothrix concilii [9,11,57] or Methanospirillum hungatei [10,12,35] 40,000 to 200,000 (75a). Heterogeneities in molecular weights can be due to proteolytic cleavage in vivo or in vitro (4,36,63) or to differences in the degree of glycosylation (39).…”

S layer regeneration in Methanococcus voltae protoplasts

“…(1991) using Methanocorpzlsczlhm sinese as a cell model. The dynamic assembly of the S layer on cell surfaces during growth is little understood, although theoretical considerations dictate that the S layer must incorporate new material in the lattice at discrete sites which act as growth points (Harris & Scriven, 1970;Messner et al, 1986;Pum e t al., 1991).…”

Methanolacinia gen. nov., incorporating Methanomicrobium paynteri as Methanolacinia paynteri comb. nov.

“…Micrographs of thin sections or freeze-etched cell preparations were taken on a Philips EM 301 electron microscope at 80 kV as described (15).…”