Role of Multivalent Cations in the Organization, Structure, and Assembly of the Cell Wall of Pseudomonas aeruginosa

Abstract

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...

Peptidoglycans (Mucopeptides): Structure, Function, and Variations

“…coli strains can be compelled to lyse with either lysozyme or chelating agents upon subsequent dilution of the sucrose. @G The only known [67][68][69][70][71][72] damage to gram-negative cells that is caused by chelating agents, such as ethylenediaminetetraacetate (EDTA) , is by removal of divalent cations, which is known to affect components of the outer membrane. A major part of the lipopolysaccharide is solubilized from the surface of the cell together with some proteins and phospholipids.…”

Role of autolysins in the EDTA-induced lysis of Pseudomonas aeruginosa

“…Incubation of many Gram-negative bacteria with ethylenediaminetetraacetic acid (EDTA) results in damage to surface structures, thereby leading to leakage of intracellular solutes [1] as well as sensitization of cells to lysozyme [2,3] and various bactericides [1,[4][5][6]. In the absence of lysozyme, EDTA alone has appreciable lytic ac-tivity against Pseudomonas aeruginosa [1,3,[7][8][9][10] and this effect is enhanced by tris (hydroxymethyl)aminomethane (Tris) buffers [9,11]. Investigations on the mechanism of action of EDTA have indicated that chelation of the Mg 2+, Ca 2+, and Zn 2+ ions associated with the lipopolysaccharides (LPS) of the P. aeruginosa cell walls [12] results in the loss of structural integrity and cell death [8,11,13,14].…”

“…In the absence of lysozyme, EDTA alone has appreciable lytic ac-tivity against Pseudomonas aeruginosa [1,3,[7][8][9][10] and this effect is enhanced by tris (hydroxymethyl)aminomethane (Tris) buffers [9,11]. Investigations on the mechanism of action of EDTA have indicated that chelation of the Mg 2+, Ca 2+, and Zn 2+ ions associated with the lipopolysaccharides (LPS) of the P. aeruginosa cell walls [12] results in the loss of structural integrity and cell death [8,11,13,14]. For cell lysis to occur, the peptidoglycan layer must also be compromised but since osmotic stability can be restored to EDTA-treated cells [11], this compromise must be confined to very localized areas of the pepti-doglycan.…”

“…Investigations on the mechanism of action of EDTA have indicated that chelation of the Mg 2+, Ca 2+, and Zn 2+ ions associated with the lipopolysaccharides (LPS) of the P. aeruginosa cell walls [12] results in the loss of structural integrity and cell death [8,11,13,14]. For cell lysis to occur, the peptidoglycan layer must also be compromised but since osmotic stability can be restored to EDTA-treated cells [11], this compromise must be confined to very localized areas of the pepti-doglycan. This action of EDTA appears to be unique since several other chelating agents are unable to replace it in sensitizing cells to lysozyme [3,8,15].…”

A comparative study on intrinsic fluorescence of BSA and lysozyme proteins in presence of different divalent ions from their solution and thin film conformations

“…[25] In addition, the structure of bacterial membranes can be stabilized by Ca 2+ and Mg 2+ ions by forming metal ion bridges between phosphate groups in phospholipids and other carboxylic groups in membrane proteins. [26] In our previous study, the optical emission behaviours of BSA and lysozyme protein molecules from bulk and thin film geometries in the presence of monovalent, divalent or trivalent ions were explored. [27] It was found that the protein-ion interactions depended on the charge of the cations and, as a result, their fluorescence emission behaviour was modified.…”