On the Peptidoglycan of the Cell Walls of Pseudomonas aeruginosa

Abstract

The peptidoglycan from cell walls of Pseudomonm aeruginosa consists of a macromolecular bag-shaped network of covalently linked repeating units. Metal ions obviously do not participate in conserving its structure, nor could any other component besides glutamic acid, 2,5-diaminopimelic acid, alanine, glucosamine and muramic acid (the latter two possibly being acetylated) be shown to be a constituent of the peptidoglycan sacculus.Approximately one out of four diaminopimelate residues of the repeating units of the peptidoglycan is cross-linked to another unit.The protein covalently bound to the peptidoglycan can be removed from the latter by proteolytic digestion. Lysis experiments proved that Tris buffer and EDTA attack the outer layers of the cell wall which protect the peptidoglycan against being hydrolyzed by lysozyme.The cell wall of gram-negative bacteria represents a very rigid structure, the form of which is maintained by a bag-shaped peptidoglycan macromolecule, the so-called sacculus The work presented here is to show that: a) no other components but those mentioned above could be shown to constitute the peptidoglycan of Ps. aeruginosa ; b) the entire peptidoglycan of one single cell wall is to be regarded as one covalently bound structure ; c) the lipopolysaccharide is not covalently linked to the peptidoglycan and can be resolved from the peptidoglycan merely by the action of detergents ; d) the covalently bound protein moieties are bound to a fraction of the subunits of the peptidoglycan by a trypsinlchymotrypsin-sensitive linkage. MATERIALS AND METHODSThe Pseudoinonus aeruginosa strain OSU 64 was provided by Dr Eagon (Athens, Ga.). Amino acid analyses were performed on a BioCal BC 200 amino acid analyzer. Atomic absorption analyses were carried out with a model 403 Perkin Elmer atomic absorption spectrophotometer. Cells were disrupted by a Biihler cell fractionator using glass beads. Electrophoreses were run on an electropherograph model "Frankfurt" (Hormuth & Vetter, Wiesloch). Cell suspensions in dodecylsulfate were homogenized by means of an Ultra-Turrax (Jahnke & Kunkel, Staufen). All reagents used were analytical grade. Preparation of the PeptidoglycanThe cells were harvested after having reached the stationary phase. Following centrifugation cells were resuspended in ten times their weight of a 40/, solution of sodium dodecylsulfate in water with the homogenizer. After standing for 10min, the homogenation was repeated three times with intermediate standing. The suspension was then centrifuged (20 min, 8000 xg). The sediment was resuspended in 4O/, sodium dodecyIsulfate and the suspension heated for 30 min on a boiling water bath and centrifuged. The sediment was suspended in 0.02 M NaHCO, and dialyzed against 2~ 10 1 of the same buffer, then twice with deionized water. The sediment collected by centrifugation of the dialyzed suspension will be called "cell walls'' hereafter. To remove the covalently bound protein, the cell walls were incubated overnight with trypsin/chymotrypsin (100 pg/ml each...

Penicillin-Resistant Mechanisms in Pseudomonas aeruginosa : Effects of Penicillin G and Carbenicillin on Transpeptidase and d -Alanine Carboxypeptidase Activities

“…The peptidoglycan isolated from cell walls of P. aeruginosa consists of macromolecular network of covalently linked repeating units (3,10). This structure of peptidoglycan is similar to that of penicillin-susceptible organisms previously reported (2,15,17).…”

“…3). (3) reported that the peptidoglycan from cell walls of P. aeruginosa consists of a macromolecular bagshaped network of covalently linked repeating units. That is to say, the peptidoglycan builds up polyacetylhexosamine and peptide chains.…”

Formation and Isolation of Leucocidin from Pseudomonas aeruginosa

“…Although maximal lysis of P. aeruginosa strain 158 with tris (pH 9)-EDTA alone took about 3 h, added lysozyme (50 pg ml-l) caused immediate lysis (Heilmann, 1972). High-speed centrifugation (40000 g, 60 min) of the jelly-like mass yielded only a small quantity of supernatant.…”

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

“…Some strains are resistant to many antibiotics, and therefore P. aeruginosa is of significant clinical interest (61)(62)(63)(65)(66)(67). The peptidoglycan of P. aeruginosa is of the A1␥ chemotype, the same as E. coli (65,(67)(68)(69), and is characterized by a 4-3 cross-link involving the stem peptide diaminopimelic acid (24). However, a quantitative analysis of the chemical composition of P. aeruginosa peptidoglycan has not been undertaken, particularly in relationship to pathogenicity.…”