SUMMARY Bacteria can respond to adverse environmental conditions by drastically reducing or even ceasing metabolic activity. They must then determine that conditions have improved before exiting dormancy, and one indication of such a change is the growth of other bacteria in the local environment. Growing bacteria release muropeptide fragments of the cell wall into the extracellular milieu, and we report here that these muropeptides are potent germinants of dormant Bacillus subtilis spores. The ability of a muropeptide to act as a germinant is determined by the identity of a single amino acid. A well-conserved, eukaryotic-like Ser/Thr membrane kinase containing an extracellular domain capable of binding peptidoglycan is necessary for this response, and a small molecule that stimulates related eukaryotic kinases is sufficient to induce germination. Another small molecule, staurosporine, that inhibits related eukaryotic kinases blocks muropeptide-dependent germination. Thus, in contrast to traditional antimicrobials that inhibit metabolically active cells, staurosporine acts by blocking germination of dormant spores.

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...

The nitrogen regulatory (NtrC) protein of enteric bacteria, which binds to sites that have the properties of transcriptional enhancers, is known to activate transcription by a form of RNA polymerase that contains the NtrA protein (sigma 54) as sigma factor (referred to as sigma 54-holoenzyme). In the presence of adenosine triphosphate, the NtrC protein catalyzes isomerization of closed recognition complexes between sigma 54-holoenzyme and the glnA promoter to open complexes in which DNA in the region of the transcription start site is locally denatured. NtrC is not required subsequently for maintenance of open complexes or initiation of transcription.

SPOR domains are ϳ70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteinsDamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus, localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.

Peptidoglycan was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains. Digestion with muramidase resulted in cleavage of the glycosidic bonds adjacent to muramic acid replaced by peptide or alanine side chains but not the bonds adjacent to muramic lactam. Reduction of the resulting muropeptides allowed their separation by reversed-phase high-pressure liquid chromatography. The structures of 20 muropeptides were determined by amino acid and amino sugar analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In wild-type spores, 50% of the muramic acid had been converted to the lactam and 75% of these lactam residues were spaced regularly at every second muramic acid position in the glycan chains. Single L-alanine side chains were found on 25% of the muramic acid residues. The remaining 25% of the muramic acid had tetrapeptide or tripeptide side chains, and 11% of the diaminopimelic acid in these side chains was involved in peptide cross-links. Analysis of spore peptidoglycan produced by a number of mutants lacking proteins involved in cell wall metabolism revealed structural changes. The most significant changes were in the spores of a dacB mutant which lacks the sporulation-specific penicillinbinding protein 5*. In these spores, only 46% of the muramic acid was in the lactam form, 12% had L-alanine side chains, and 42% had peptide side chains containing diaminopimelic acid, 29% of which was involved in cross-links.Bacterial endospores produced by gram-positive species (including Bacillus and Clostridium species) are metabolically dormant resting-stage cells resistant to a variety of physical and chemical treatments which are rapidly lethal to vegetative cells. These resistance properties derive from a variety of modifications in the cell structure and contents. The major determinant of spore heat resistance has been found to be the relative degree of dehydration of the spore protoplast (1,14,17,18). The relative dehydration of the protoplast is maintained by a surrounding peptidoglycan structure. This structure is composed of a thin inner layer called the germ cell wall and a thicker outer layer termed the cortex. The germ cell wall has a structure similar to that of the vegetative wall peptidoglycan (27) and serves as the precursor of the vegetative wall upon spore germination. The cortex peptidoglycan has a significantly different structure (30, 32) (Fig. 1), with the most dramatic changes being the absence of side chains from approximately 50% of the muramic acid residues which are converted to muramic lactam and the presence of single L-Ala side chains on a large fraction of the muramic acid residues.Degradation of the cortex peptidoglycan, during spore germination or by artificial means, results in rapid rehydration of the protoplast and a concomitant loss of spore heat resistance and dormancy. Several theories suggest that the cortex peptidoglycan also has a mechanical activity that is involved in the attainment of spore protoplast dehy...