In vitro synthesis and O acetylation of peptidoglycan by permeabilized cells of Proteus mirabilis

Abstract

The synthesis and 0 acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-j-_4CjN-acetyl-Dglucosamine was found to be radioactive. The peptidoglycan (PG) of Proteus mirabilis is similar in composition and structure to those of other members of the family Enterobacteriaceae, except for one important distinction, the presence of 0-linked acetyl groups. This modification to PG occurs at the C-6 hydroxyl group of N-acetylmuramyl residues, producing the corresponding 2,6-diacetylmuramyl derivative. O-Acetylated PG is resistant to the hydrolytic activity of many lysozymes, including human ones (11 and references therein). Thus, following infection, large fragments of 0-acetylated PG persist and circulate in a host organism, enhancing the induction of the many pathobiological effects of PG (for a review, see reference 30), including rheumatoid arthritis (14). That a number of bacteria, including many pathogenic species (both gram-positive ones, e.g., Staphylococcus aureus [16,31,35], and gram-negative ones e.g., Neisseria gonorrhoeae [34] and P. mirabilis [11,13]) possess 0-acetylated PG makes this an important phenomenon that ironically has received little attention. The PG of some species of bacteria has been reported to be 0 acetylated to the extent of up to 70%, so that it can confer both intrinsic resistance and complete resistance to lysozyme hydrolysis (4,11,20,25,27).Although 0-acetylated PG was first observed more than 30 years ago (1, 7) and the biological significance of this modification was discerned soon after (6), very little is known about the biosynthetic process involved in PG 0 acetylation. It is known to be stimulated either when cells are in the stationary phase of growth or under conditions which mimic the stationary phase (e.g., under the influence of chloramphenicol [28] acetylation occurs within the PG sacculus and outside the cytoplasm (9,17,18,(21)(22)(23)33). Indeed, PG 0 acetylation has also been demonstrated to continue in an in vitro P. mirabilis PG biosynthetic system (25), albeit to a lesser extent than that observed in vivo. Furthermore, searches for lipid(bactoprenyl)-linkedN-acetylglucosaminyl-N,O-diacetylmuramyl-pentapeptide precursors in either the cytoplasm or the cytoplasmic membrane have proved futile (22,33). These observations thus suggest that both an acetyltransferase and a source of transferable acetate must be present outside the cytoplasm. Such conditions would preclude the utilization of the typical activated acetate precursors, such as acetyl coenzyme A or acetyl phosphate, since they are not transported out of the cytoplasm.Through in...

Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan

“…For N-acetyltransferases, the employed cosubstrate is typically [acetyl- 14 C]CoA, but its substitution with either [acetyl-3 H]GlcNAc or radiolabelled peptidoglycan fragments (specifically labelled at the N-2 and/or O-6 position of both N-acetylglucosaminyl and N-acetylmuramyl residues) led to the recovery of radioactive filters. This activity of AAC(2Ј)-Ia from P. stuartii was observed both in vivo and in vitro.…”

O-Acetylation of Peptidoglycan in Gram-negative Bacteria

“…Cell-free Assay for PG O-Acetylation-The toluene-based permeabilization method for preparing cell-free fractions of wall components of P. mirabilis and assay for PG O-acetylation described previously (27,28) was adapted for use with E. coli Top10 transformants harboring pBAD18-Cm, pBADHis-A, pACPM17, or pACPM19. Immediately following toluene treatment, 10 ml of E. coli cells expressing ape2, as well as control cells containing an empty pBAD18-Cm or pBADHis-A vector, in 100 mM Tris-HCl buffer, pH 6.8 containing 30 mM MgCl 2 were incubated with 15 mM acetyl-CoA at 30°C for 30 min.…”

Contribution of gentamicin 2'-N-acetyltransferase to the O acetylation of peptidoglycan in Providencia stuartii

“…Substantial evidence indicates that the O acetylation of peptidoglycan occurs after the cross-linking of peptide side chains by transpeptidation (4,12,17,18,(25)(26)(27)36), and more recently, an N3O-acetyltransferase has been proposed to catalyze the transfer of acetyl groups from the N-2 position of either glucosaminyl or muramyl residues to the C-6 position of the latter during the turnover of peptidoglycan (14,15). In the present study, we have investigated the relationship between the expression of the aac(2Ј)-Ia gene in P. stuartii and the O acetylation of its peptidoglycan.…”