Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation

Abstract

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the ␦-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid ␦-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% ␦-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulationspecific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid ␦-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of ␦-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed. Dormant bacterial endospores formed by the genera Bacillus and Clostridium are the most resistant living structures known and are able to survive thousands if not millions of years (9). During the quiescent state, spores exhibit high-level resistance to many treatments, including heat, UV light, desiccation, and the action of deleterious chemicals. As a result of their resistance properties, spores are able to survive many food preservation and pasteurization procedures and thus cause huge problems to the food industry (11).Endospores are characterized by a relatively dehydrated protoplast encased in integument layers (20). The most prominent of the integuments are the spore coat layers which determine the physical properties of the spore surface and are responsible for resistance to enzymatic assault (56). However, the spore coats are not involved in the maintenance of dormancy and heat resistance (56). Between the spore coats and the protoplast membrane is a thick layer of bacterial peptidoglycan, consisting of two sublayers. Innermost is the ...

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

“…To test the contribution of DPA to GFP immobility we used spores carrying both the gfp fusion and a cwlD mutation. This latter mutation results in a modified spore cortex lacking muramic acid lactam, the recognition determinant for the enzymes that initiate cortex hydrolysis during spore germination (2,8,36,37). Consequently, although cwlD spores will initiate germination in response to nutrients and release DPA rapidly, they cannot progress past stage I of germination, and the core water content of germinated cwlD spores [Ϸ60% of wet weight (7,8)] is only slightly above that of dormant spores of several other Bacillus species (5).…”

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

“…The Bacillus endospores are characterized by a relatively dehydrated protoplast encased in integument layers, including spore coat, cortex, and primordial cell wall, which endow the spores with resistance to a variety of harsh treatments, such as heat, UV light, desiccation, and toxic chemicals [40]. Endospores could barely be transformed, and thus sporulation rate is one of the crucial factors for transformation.…”

Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination

“…Spores were prepared from cultures allowed to sporulate in CCY medium (Stewart et al, 1981) as previously described (Atrih et al, 1996). Purified spores were heat-activated at 70 mC for 30 min and cooled in ice before the addition of germinants.…”

“…Preparation of peptidoglycan from spore cortex and vegetative cells of B. subtilis, muropeptide separation by RP-HPLC, and amino acid analysis were performed as described by Atrih et al (1996Atrih et al ( , 1998.…”