ASBELL, MARY A. (University of Georgia, Athens), AND R. G. EAGON. Role of multivalent cations in the organization, structure, and assembly of the cell wall of Pseudomonas aeruginosa. J. BacterioL 92:380-387. 1966.-Incubation of Pseudomonas aeruginosa with ethylenediaminetetraacetate induced the formation of osmotically fragile rods termed osmoplasts. These could be restored to osmotically stable forms by multivalent cations. Only those cells restored by divalent cations normally found in the cell wall were capable of multiplication. The respiration of restored cells, however, was unimpaired, irrespective of whether they were capable of multiplication. Moreover, the permeability characteristics of osmoplasts and restored cells were unimpaired. When multivalent cations were chelated from the cell wall and replaced by sodium, a weakened cell wall and an osmotically fragile cell resulted. This was apparently caused by the absence of cross-linkages in the cell wall via multivalent cations. Tris(hydroxymethyl)aminomethane buffer compounded the lethal effects of ethylenediaminetetraacetate. The lipopolysaccharide component was inferred to be the site of attack by ethylenediaminetetraacetate. A mechanism for the synthesis of the lipopolysaccharide sacculus was proposed whereby negatively charged subunits are "trapped" by forming ionic and coordinate bonds intermediated by multivalent cations. Recently, Eagon and Carson (7) and Gray and Wilkinson (10) reported that Pseudomonas aeruginosa dies rapidly in the presence of ethylenediaminetetraacetate (EDTA). Evidence was presented by both groups that the structural integrity of the cell wall is impaired The former authors concluded, moreover, that the binding of divalent cations is essential for the integrity of the cell wall of this microorganism Furthermore, Eagon, Simmons, and Carson (8) reported that Ca++, Mg++, and Zn++ are components of the cell wall of P. aeruginosa. On the other hand, evidence was presented by Carson and Eagon (6) that the mucopeptide component is not solely responsible for the structural integrity of the cell wall of P. aeruginosa. The investigations described in this paper, therefore, were undertaken to provide further information on the role of multivalent cations in the organization and structure of the cell wall of P. aeruginosa. A role for multivalent cations in the nonenzymatic assembly of subunits of lipopolysaccharide or lipoprotein components of the cell wall, or both, was inferred as well from results of our investigations. A preliminary report of this work has been published (2). MATERIALS AND METHODS Cultivation of organism. P. aeruginosa strain OSU 64 was cultivated at 37 C on a rotary shaker in a basal salts-glucose-yeast extract medium as previously described (6). Cells were harvested after 14 to 16 hr of cultivation and were washed with distilled water before use. Experimental procedures. Cells of P. aeruginiosa were incubated with EDTA, tris(hydroxymethyl)aminomethane (Tris) buffer, and lysozyme according to techniques previously rep...