The response of selected members of the archaea to the Gram stain

Abstract

NIG 2W1Archaea possess a broader range of cell envelope structural formats than eubacteria and their cell walls do not contain peptidoglycan. Some archaea have only a single S-layer as their cell wall (e.g. Methanococcus jannaschii and Sulfolobus acidocaldarius), whereas others have multiple layers (e.g. Methanospirillum hungatei). Sometimes there can also be a high proportion of tetraether lipids in membranes to make the envelope more resilient to environmental stress (e.g. Methanococcus jannaschii and Sulfolobus acidocaldarius grown a t 70 "C). Since the Gram reaction depends on both the structural format and the chemical composition of the cell envelope of eubacteria, it was important to determine if the same is true for archaea. Methanospirillum hungatei, Methanosarcina mazei, Methanobacterium formicicum , Methanococcus jannaschii and Sulfolobus acidocaldarius, chosen because of their different envelope formats and chemistries, were subjected to a Gram stain that can be used for transmission electron microscopy. In this staining regimen, the iodine is replaced by potassium trichloro($-ethylene)platinate(lI) as the mordant, and the platinum of the new compound is the electron-scattering agent for electron microscopy. Of al I these archaea, only Methanobacterium formicicum stained Gram-positive since its pseudomurein wall remained intact; the platinum compound formed large electron-dense aggregates with the crystal violet that were located in the vicinity of the cell wall and the plasma membrane. All but the terminal filament cells of Methanospirillum hungatei stained Gram-negative because the limiting porosity of its external sheath was so small that the Gram reagents could not enter the cells. The terminal cells of filaments stained Gram-positive because the staining reagents gained entry through the terminal plugs. All other archaea stained Gram-negative because their cell walls were so disrupted during staining that the crystal violet-platinum complex could not be retained by the cells. Methanococcus jannaschii was grown a t both 50 "C and 70 "C so that the tetraether lipids in its plasma membrane could be increased from 20% (50 "C) to 45% (70 "C) of the total lipids; in both cases the cells stained Gram-negative.

Mechanism of osmoprotection by archaeal S-layers: A theoretical study

“…I, the protein connections per unit cell of the sheath must resist lateral forces of 18-40 nN. On the other hand, studies with Methanococcus jannaschii, an Euryarchaeon possessing an Slayer of the Crenarchaeota type (Beveridge and Schultze-Lam, 1996), showed that cells grown at 260·10 5 Pa gas pressure were ruptured if decompression was too fast, but cells taken from cultures at 7.8·10 5 Pa remained intact (Park and Clark, 2002), which is in agreement with the quantitative assessment outlined above. The turgour of various Gram-negative bacteria is in the range of ≈ 1·10 5 -6·10 5 Pa as determined by means of the gas vesicle approach (Koch and Pinette, 1987;Beveridge, 1988;Overmann et al, 1991).…”

V. Functions of S-layers

“…An energy dispersive X‐ray spectrum of an uncontrasted thin section is shown in the inset and the TPt precipitates are identified by the Pt peaks (arrows). (This figure previously appeared in Microbiology [9]in an article by the author.)…”

“…Both of these simple S‐layered bacteria stained Gram‐negative and this was independent of growth temperature or growth phase [9]. Thin sections of them showed little cell substance to be left after the TPt‐Gram stain (Fig.…”

“…4), these archaeal S‐layers were not able to stand up to the rigors of the Gram stain. Even when there was a substantial increase of tetraether lipid in the plasma membrane, the cells still lysed and stained Gram‐negative [9].…”

Identification of regulated genes conferring resistance to high concentrations of glyphosate in a new strain ofEnterobacter

“…To identify the sample, Gram stain tests were performed under light microscopy (Beveridge & Schultze-Lam, 1996). Transmission electron microscopy (TEM) studies were also performed: bacteria were cultivated to an OD 600 nm of 0.6-0.8, centrifuged (1500 g for 5 min at 4°C), and the supernatant washed twice with phosphatebuffered solution (PBS) before samples were immediately fixed with 2.5% (v/v) glutaraldehyde prepared in PBS.…”