The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.The gram-positive, gut commensal bacterium Enterococcus faecalis is the causative agent of 90% of all enterococcal infections, which include endocarditis, bacteremia, and urinary tract infections. Of these, urinary tract infections are the most common, and the majority of them are acquired nosocomially. E. faecalis infections pose problems for the clinician because of their resistance to multiple antibiotics, sometimes including vancomycin, the drug of last resort for many gram-positive infections (recently reviewed in references 14, 19, and 32). To exacerbate this situation, this bacterium has been shown to persist in a mouse model for extended periods of time in the kidney (26). This persistence is thought to result from the cell's ability to enter the viable but nonculturable (VBNC) state, a feature first described for E. faecalis by Lleo et al. (30). VBNC E. faecalis, like other VBNC pathogenic bacteria, appears to retain its pathogenicity genes and is able to resume active growth upon restoration of the optimal environmental conditions (reference 30 and references therein). While in this state, E. faecalis cells have an irregular morphology, and there are concomitant alterations in the molecular architecture of the peptidoglycan sacculus (39). However, the changes observed in the peptidoglycan composition do not readily account for the persistence associated with the VBNC enterococci. One possible explanation for the apparent resilience of VBNC E. faecalis could involve a modification to the pep...
The gentamicin 2'-N-acetyltransferase [EC 2.3.1.59; AAC(2')-Ia] of Providencia stuartii was shown to contribute to the O-acetylation of peptidoglycan and mutants that either under- or overexpress the aac(2')-Ia gene was characterized phenotypically to possess either lower or higher levels of peptidoglycan O-acetylation, respectively, compared to the wild-type. These mutants were subjected to scanning electron microscopy. P. stuartii PR100, with 42-44% peptidoglycan O-acetylation compared to 54% for the wild-type, appeared as irregular rods. In direct contrast, strains PR50.LM3 and PR51, with increased levels of peptidoglycan O-acetylation (63 and 65%, respectively), appeared as coccobacilli or chain formers, respectively. Zymogram analysis of the autolysins produced by another member of the closely related Proteeae group of bacteria, Proteus mirabilis, indicated the presence of three classes of enzymes: one that acts preferentially on native, O-acetylated peptidoglycan, a second that hydrolyses non-O-acetylated peptidoglycan, and a third that is not distinguished by the two forms of substrate. On the basis of the apparent morphological changes directly related to levels of O-acetylation combined with the presence of different classes of autolysins, a model is proposed that invokes the role of this modification in the control of autolysins for the maintenance of the structure of the peptidoglycan sacculus.
The O-acetylation of peptidoglycan in Gram-negative bacteria occurs specifically at the C-6 hydroxyl group of muramoyl residues. The level of peptidoglycan O-acetylation was found to decrease from 51% to 29% upon differentiation of Proteus mirabilis vegetative cells to swarmers. This decrease was accompanied by a change in the muropeptide composition of the peptidoglycan. In particular, the content of anhydromuropeptides increased, while the amount of Lys-Lys-muropeptides arising from bound lipoprotein decreased. These changes together with a shift in proportion of larger muropeptides suggested a decrease in average chain length of the muropeptides from swarmer cells. Zymography using SDS-PAGE gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the activity of specific autolysins during the differentiation of vegetative to swarming cells of P. mirabilis. A 43 kDa autolysin with increased specificity for O-acetylated peptidoglycan was detected in vegetative cells, but its activity appeared to decrease as the cells began to differentiate, while the levels of 3 other autolysins with apparent specificity for non-O-acetylated peptidoglycan increased. These changes are discussed in relation to the autolysin profile of the bacteria and the changes in peptidoglycan composition with cell differentiation.
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