Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores

Abstract

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant L-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic delta-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.

FIG. 1

Analysis of muropeptides by RP-HPLC during germination (120 min) of B. subtilis 168 HR spores. Muropeptide-containing samples were separated by RP-HPLC, and the A₂₀₂ of the eluates was monitored. (A) Dormant spore-associated material; (B) germinated spore-associated material; (C) germination exudate; (D) germination exudate (no Cellosyl digestion or reduction).

FIG. 2

Portions of the ROESY spectra of the corresponding dormant and germination-associated tetrasaccharide alanine muropeptides 11 and G4 (a and b, respectively). The spectra show nuclear Overhauser enhancements between the 2′-amide protons (and alanine amide proton) and other protons in the muropeptides. The protons are labeled at the top with the identity of the saccharide unit (from A at the nonreducing end to D at the reducing end). Chemical shift assignments for these muropeptides are given in Table 3.

FIG. 3

Positive (A)- and negative (B)-ion MALDI mass spectrum of muropeptide G12 (Tables 1 and 2; anhydro-hexasaccharide tetrapeptide) obtained in the reflector mode.

FIG. 4

Kinetics of biochemical events during germination of B. subtilis 168 HR spores. •, percent loss of heat resistance; ○, percent loss of A₆₀₀; ■, amount of muropeptide G3; □, amount of muropeptide G4.

FIG. 5

Differential muropeptide release during germination of B. subtilis 168 HR. Amounts are calculated as a percentage of the dormant spore value. ○, hexasaccharide-containing muropeptides; •, tetrasaccharide-containing muropeptides; ■, disaccharide-containing muropeptides with alanine or tetrapeptide side chains; □, disaccharide-containing muropeptides with tripeptide side chains.