Isolation and Study of the Composition of a Peptidoglycan Complex Excreted by the Biotin‐Requiring Mutant of Brevibacterium divaricatum NRRL‐2311 in the Presence of Penicillin

Abstract

When cultures of biotin-requiring mutant of Brevibacterium divaricutum NRRL-2311 were treated in early logarithmic phase with penicillin (2 -3 units/ml) in a glucose-mineral medium under excess supply of biotin, large amounts of a high-molecular-weight material accumulated in the medium. The phenomenon could not be observed with cultures run in parallel from which penicillin was omitted. Chemical analyses of the excreted material isolated from the media of I-h and 24-h penicillin-treated cultures, showed that the main constituents were peptidoglycan components of noncross-linked structure bearing both L-and D-alanine residues ; evidence was also obtained for the occurrence of extractable lipid material, non-amino sugars and organic phosphate.Under identical conditions, the excretion of peptidoglycan could be induced by ampicillin and cloxacillin, respectively, but not by bacitracin. Addition of penicillin to biotin-requiring mutant of M . glutamicus yielded similar results, indicating that the phenomenon was not restricted to the Brev. divaricutum strain. This suggests that excretion of peptidoglycan material by the two biotin-requiring mutants might be the result of two events, (a) a change in the osmotic barrier of the cell and (b) specific inhibition of cross-link formation in peptidoglycan induced by penicillin.Several procedures were examined for the isolation and purification of the peptidoglycan complex excreted by penicillin-treated Brev. mutant. Suitable labelling experiments with L-[U-14C]glutamic acid and analyses of lysozyme digests of the purified fractions, suggest that some of the components might contain murein fragments covalently linked to a polysaccharide portion.During our studies on the accumulation of amino acids into the culture broth of the biotin-requiring mutant of Brevibacteriurn divaricatum (NRRL-231 l ) , it has been observed that in the presence of low doses of penicillin the extracellular accumulation of glutamate is accompanied by the excretion of some highmolecular-weight material. The first observations were made in cultures grown on glucose media containing biotin, to which penicillin G (2-10 units/ml) was added at the early logarithmic phase; incubations were carried out at 28 "C for 24, 48 and 72 h, respectively.When a growing culture was incubated simultaneously with 14C-labelled L-glutamic acid and penicillin for 24 h and the supernatant fluid submitted to gel fractionation, a substantial portion of radioactivity eluted a t low retention volumes. I n contrast, in the culture fluid of the control from which penicillin had been omitted, no radioactivity associated with some high-molecular-weight material could be detected. When subjected to paper electrophoresis and chromatography, the radioactive material excreted by penicillin-treated cells did not move from the origin. Hydrolysis of a sample in 6 M HCl a t 100 "C revealed diaminopimelic acid as one of the amino acid components.Since diaminopimelate is present in no other cellular material than in murein, the excreted mater...

Effect of peptidoglycans on erythrocyte survival

“…The preparation was made as previously described (Rosenthal and Dziarski, 1994). Briefly, Gram-positive bacteria grown in the presence of b-lactam antibiotics secrete soluble polymeric PGN fragments (Barrett and Shockman, 1984;Fischer and Tomasz, 1984;Keglevic et al, 1974;Mirelman et al, 1974;Tynecka and Ward, 1975;Zeiger et al, 1982). Isolation of this soluble PGN includes (i) obtaining exponentially growing cells in enriched medium; (ii) harvesting the cells and transferring them into synthetic medium; (iii) growing the cells in the synthetic medium in the presence of penicillin; (iv) obtaining and concentrating the PGN-containing supernatant; and (v) isolating and purifying soluble PGN from the supernatant.…”

Immunomodulatory activity of novel adjuvant formulations based on Montanide ISA oil-based adjuvants and peptidoglycan monomer

“…PGM was from Pliva, Croatia [4,17]. Complete and Incomplete Freund's adjuvants (CFA, IFA) were purchased from Sigma, USA.…”

“…PGM is an adjuvant active molecule with a potential for use in human and veterinary vaccine. It is water-soluble, non-immunogenic, non-toxic and non-pyrogenic compound of bacterial origin with completely defined chemical structure GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(εNH 2 )-D-Ala-D-Ala [4,5]. Adjuvant effects of PGM have been demonstrated in mice model using different types of antigens [6][7][8][9].…”

Immunotherapy of lewis lung carcinoma with hydrosoluble peptidoglycan monomer (PGM)

“…PGM was prepared by lysozyme digestion of uncrosslinked peptidoglycan chains isolated from culture fluids of penicillin-treated Brevibacterium divaricatum NRRL-2311, as described in detail elsewhere [11,12]. PGM was tested by the limulus amebocyte lysate (LAL) test (Pyrostat kit: Millipore, New Jersey, USA), and the samples used in the present study were found to contain 0.015 ng endotoxin per mg PGM.…”

“…PGM is a hydrosoluble substance originating from the cell wall of Brevibacterium divaricatum [11,12], and is devoid of toxic effects for the host in the range of doses having immunostimnlating activity [6]. The differential effects of PGM on primary tumor growth and on the formation of spontaneous lung metastases have thus been examined.…”