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

Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1

¹Author for correspondence: Terry J. Beveridge. Tel: +1 519 824 4120 ext. 3366. Fax: +1 519 837 1802. e-mail: tjb@micro.uoguelph.ca

ABSTRACT

Archaea 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 at 70 ⁰C). 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(²-ethylene)platinate(II) as the mordant, and the platinum of the new compound is the electron-scattering agent for electron microscopy. Of all 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 at both 50 ⁰C and 70 ⁰C so that the tetraether lipids in its plasma membrane could be increased from 20% (50 ⁰C) to 45% (70 ⁰C) of the total lipids; in both cases the cells stained Gram-negative.

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