Analysis of Peptidoglycan Structure from Vegetative Cells of Bacillus subtilis 168 and Role of PBP 5 in Peptidoglycan Maturation

Abstract

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilisvegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in thedacA mutant. This major low-molecular-weight PBP (dd-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

AcmA of Lactococcus lactis is an N‐acetylglucosaminidase with an optimal number of LysM domains for proper functioning

“…These peaks were compared with those obtained by hydrolysis of Bacillus peptidoglycan with the muramidase Cellosyl (Fig. 5C) [21]. The AcmA-specific peaks were not identical to the major muramidase peaks.…”

“…Determination of the hydrolytic activity of AcmA using RP-HPLC analysis B. subtilis peptidoglycan was isolated as described previously [21]. Purified peptidoglycan (10 mg) was incubated with partially purified AcmA in 1 mL 50 mm KP i buffer (pH 5.5) and incubated overnight at 37°C.…”

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

“…In L. monocytogenes, cells lacking PBP5 displayed an irregular morphology and shape (Guinane et al, 2006), and a thicker cell wall (Korsak et al, 2005b). Moreover the ratio of pentapeptides to tripeptides was increased in cells lacking PBP5 (Korsak et al, 2005b), as in a dacA (PBP5) mutant of B. subtilis (Atrih et al, 1999). In Streptococcus pneumoniae, PBP3 plays a major role in organizing the growth and division process (Morlot et al, 2004).…”

Bacterial peptidoglycan (murein) hydrolases

“…DacB is expressed only in the mother cell compartment of the developing sporangium whereas dacF is expressed in the forespore compartment and they can act differently on the nascent spore peptidoglycan (Popham et al, 1999). The proportion of pentapeptides was largely increased in a dacA (PBP5) mutant of B. subtilis (Atrih et al, 1999). In L. monocytogenes, cells lacking PBP5 displayed an irregular morphology and shape (Guinane et al, 2006) and a thicker cell wall (Korsak et al, 2005a).…”

“…Then, PdaA deacetylates the N-acetyl muramic acid product and forms the lactam ring via a cyclization event (Fukushima et al, 2002;Gilmore et al, 2004). The spore cortex is the least cross-linked of any peptidoglycan, at only 2.9% of muramic acid residues (Atrih et al, 1996), compared with that seen in vegetative cell walls, 29-33% (Atrih et al, 1999). A gradient of cross-linking across the cortex may allow inward mechanical pressure, maintaining a decreased volume and water content of the spore core and thus dormancy (Popham et al, 1999;Meador-Parton & Popham, 2000).…”