Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance

Abstract

Bacterial endospores derive much of their longevity and resistance properties from the relative dehydration of their protoplasts. The spore cortex, a peptidoglycan structure surrounding the protoplasm, maintains, and is postulated to have a role in attaining, protoplast dehydration. A structural modification unique to the spore cortex is the removal of all or part of the peptide side chains from the majority of the muramic acid residues and the conversion of 50% of the muramic acid to muramic lactam. A mutation in the cwlD gene of Bacillus subtilis, predicted to encode a muramoyl-L-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination͞outgrowth process to produce viable cells. Addition of germinants resulted in the triggering of germination with loss of spore refractility and the release of dipicolinic acid but no degradation of cortex peptidoglycan. Germination in the presence of lysozyme allowed the cwlD spores to produce viable cells and showed that they have normal heat resistance properties. These results (i) suggest that a mechanical activity of the cortex peptidoglycan is not required for the generation of protoplast dehydration but rather that it simply serves as a static structure to maintain dehydration, (ii) demonstrate that degradation of cortex peptidoglycan is not required for spore solute release or partial spore core rehydration during germination, (iii) indicate that muramic lactam is a major specificity determinant of germination lytic enzymes, and (iv) suggest the mechanism by which the spore cortex is degraded during germination while the germ cell wall is left intact.The ability of endospores produced by Gram-positive eubacteria (i.e., Bacillus and Clostridium) to survive harsh physical and chemical treatments and to remain in a dormant state for long periods is important in the food preservation industry and represents an interesting biological problem. The protoplast of the spore is relatively dehydrated (even when suspended in H 2 O) in comparison to that of a vegetative cell, resulting in metabolic dormancy, and this dehydration is responsible in large part for the heat and hydrogen peroxide resistance properties of the spore (1-3). The mechanisms by which this dehydration is achieved and maintained are not clear and remain an outstanding question in the field. Maintenance of protoplast dehydration is clearly dependent on the integrity of a surrounding peptidoglycan wall and the unique structure of this peptidoglycan has been postulated to confer on it a role in attaining protoplast dehydration (4, 5). The spore peptidoglycan is comprised of two contiguous structures, an inner layer called the germ cell wall and a thicker outer layer called the cortex. The germ cell wall appears to have a structure similar to that of vegetative cell wall peptidoglycan (6) and serves as the initial cell wall during spore outgrowth. The vegetative peptidoglycan of Bacillus subti...

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

“…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). As was found with GFP in dormant wild-type spores, GFP in dormant cwlD spores was also essentially immobile ( Fig.…”

“…Subsequently, the large peptidoglycan cortex around the spore core is degraded, and removal of this restraint allows the spore core to expand rapidly by uptake of water and thus complete stage II of germination (2,7). This latter event results in a level of core water (75-80% of wet weight) that is similar to that in growing cells (8). As noted above, there is neither metabolism nor enzyme action in the cytoplasm of dormant spores, although there are several enzyme-substrate pairs located in this region of the spore (3).…”

Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile

“…The iosgenic Bacillus subtilis strains used in this work are derivatives of strain 168 and were: PS832, a prototrophic derivative of strain 168; PS3207, cwlD::cam, in which the cwlD gene responsible for production of muramic acid-␦-lactam in the spore cortex (14,26) has been deleted and replaced with a chloramphenicol resistance marker; and PS3328 (27), cotE::tet in which the cotE gene responsible for proper assembly of much of the spore coat as well as outer spore membrane integrity (28) has been deleted and replaced with a tetracycline resistance marker. The fluorescent probes used were pyridinium, 4-…”

“…A variety of data indicate that it is the spore's inner membrane that is the major permeability barrier restricting the passage of small molecules into the spore core (3-7), although the lipid composition of the inner membrane exhibits no anomalies that might explain its unusual properties (8)(9)(10)(11)(12). In addition, the dormant spore's inner membrane has the potential to expand significantly, because electron microscopy indicates that the volume this membrane encompasses (the spore core): (i) decreases as much as 2-fold late in sporulation (13); and (ii) increases up to 2-fold in the first minute of spore germination, when the spore's large peptidoglycan cortex is degraded and the germ cell wall expands (13)(14)(15). This increase in core volume during spore germination takes place without new membrane synthesis, because ATP production is not required, and restores relatively normal permeability to the germinated spore's plasma membrane (4,5,16,17).…”

Development of natto with germination-defective mutants of Bacillus subtilis (natto)

“…Sekiguchi et al (1995) reported that the germination rate of the cwlD mutant in B. subtilis was less than 3.7 x 10 -8 , but our results were 1.9x 10 -4 and 1.7 x 10 -3 for 168 and NAFM5, respectively. Popham et al (1996) showed that the germination rate of the cwlD mutant in B. subtilis was 5 x 10 -4 (with the respect to the wild type).…”