The degree of cross-linking of the peptidoglycan of Staphylococcus aureus H and mutants lacking penicillinbinding proteins 1 and 4 was studied. No major changes were observed in organisms lacking protein 1 whereas loss of protein 4 was accompanied by a marked reduction in the degree of cross-linking and the absence of a membrane-bound 'model' transpeptidase activity. A similar effect was achieved when cultures of the staphylococci were treated with the P-lactam antibiotic cefoxitin. At low concentrations (0.05 pg m1-l) cefoxitin shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross-linkage.The possibility that the decrease in cross-linkage was a consequence of DD-carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. uureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxitin and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown.We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross-linking of peptido-,glycan and this activity is necessary to achieve the high degree of cross-linkage observed in the peptidoglycan of staphylococci.The penicillin-binding proteins of both gram-positive and gram-negative bacteria have received considerable attention in recent years [I -31. These investigations have resulted in the general conclusion that the interaction of B-lactams with one or more of the binding proteins is responsible for the lethal effects of these antibiotics. However, only in Escherichia coli has the role of individual penicillin-binding proteins been investigated in detail. In this organism distinct penicillinbinding proteins have been implicated in the determination of cell shape [4,5] ; cell division [6] and elongation [4,5,7,8]. With the exception of penicillin-binding protein 2, all have now been isolated and purified [9-141 and in the case of protein 1A [I21 and 1B [9,10,13] shown to catalyse in vitro transglycosylation and transpeptidation reactions using lipid intermediates as the peptidoglycan precursors. The lower molecular weight proteins (4, 5 and 6) catalyse DD-carboxypeptidase and in the case of proteins 5 and 6 'model' transpeptidase activity [I 1,141. Mutants lacking these proteins (4 = ducB 5 = ducA) grow normally under various conditions and it was concluded these proteins were 'non-essential' Similar studies on gram-positive bacteria have tended to concentrate on Staphylococcus aureus and various Bacillus spp. (For review, see [2].) In the bacilli, four to six distinct penicillin-binding proteins have been described in which the one of lowest molecular weight (protein 5 in Bacillus subtilis) has DD-carboxypeptidase and 'model' transpeptidase activity [I 9,201 Enzyme. DD-Carboxypeptidase or muramoyl-pentapeptide carboxypeptidase (EC 3.4.17.8).H. A. Chase, unpublished observations). A similar situation exists in S. uur...
P-N-Acetylglucosaminidase has been purified from the walls of Bacillus subtilis 168 and compared with the other known autolysin, N-acetylmuramyl-L-alanine amidase (amidase). The /?-N-acetylglucosaminidase was a dimer in LiCl buffers with a sub-unit molecular weight of 90000 and a pH optimum of about 5.0. It was very sensitive to proteolytic enzymes and was chically activated by 0.1 to 0.2 M-LiCl. It was insoluble in concentrations of LiCl lower than 0.05 to 0.1 M. It was less strongly bound to walls than was the amidase, which was a monomer of molecular weight 30000 to 40000 in LiCl buffers. The Q-N-acetylglucosaminidase is an endoenzyme and showed no exo-activity. Lysozyme-like enzyme (muramidase) activity was undetectable in the wall extracts examined.
A cell-free membrane preparation from a poorly lytic mutant of Bacillus licheniformis was used to synthesize radioactive peptidoglycan. The product was apparently un-cross-linked. When UDP-N-acetyl[(14)C]glucosamine was used and the final peptidoglycan subjected to Smith degradation, no radioactive glycerol was found. On the other hand, when peptidoglycan labelled with meso-diamino[(14)C]pimelic acid was first hydrolysed in 0.1m-HCl at 60 degrees C for 2h and then subjected to alkaline conditions, radioactive lactyl-peptides were eliminated. The proportion of radioactive lactyl-peptide decreased with increasing time of incorporation. It is concluded that the glycan chains grow by extension at their reducing ends while remaining attached by some linkage labile to mild acid, such as a glycosyl link to undecaprenol pyrophosphate.
Teichoic acids in which glycosyl residues form part of the polymer chain ..
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