Bacterial shape and cellular resistance to cytoplasmic turgor pressure are determined by peptidoglycan (PG), a polymer of repeated subunits of an N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) peptide monomer that surrounds the cytoplasmic membrane (). Covalent interactions between the stem peptides arising from separate chains typically crosslink the GlcNAc-MurNAc polymers, although in some organisms this crossbridge is composed of one or more amino acids. Most Gram-positive bacteria contain an l-lysine residue at the third position of the stem peptide (, left), whereas Gram-negative bacteria and most endospore formers have an m-Dpm (meso-diaminopimelic acid) residue in this position (, right).
Peptidoglycan fragments serve as signals in a range of host-microbe interactions including B. pertussis
infection and V. fischeri
-squid symbiosis (Cloud-Hansen et al., 2006
). They also stimulate the innate immune response (Hasegawa et al., 2006
) by binding to host proteins like Nod1 (Girardin et al., 2003
). Peptidoglycan fragments are generated by growing cells as peptidoglycan hydrolases and amidases partially digest the mature peptidoglycan to allow insertion of additional peptidoglycan monomers (Doyle et al., 1988
). While Gram-negative bacteria can efficiently recycle the resulting muropeptides, the lack of a similar recycling system in Gram-positive bacteria results in the release of large quantities of peptidoglycan fragments into the extracellular milieu by growing cells (Doyle et al., 1988
; Mauck et al., 1971
Dormant bacteria must monitor nutrient availability so that they can reinitiate metabolism when conditions become favorable. This could be accomplished by determining changes in the levels of individual nutrients. Alternatively, the growth of other bacteria in the environment would also indicate the presence of favorable conditions. Since growing bacteria release muropeptides into the environment, these molecules could serve as an intercellular growth signal to dormant bacteria.
Some Gram-positive species produce dormant spores under conditions of nutritional limitation. These cells are resistant to harsh environmental conditions and can survive in a dormant state for years (Nicholson et al., 2000
). Spores exit from dormancy via the process of germination that is triggered by specific molecules known as germinants. Most spore-forming bacteria encode several germination receptors; for example, the B. subtilis
GerAA/AB/AC proteins are necessary for germination in response to L-alanine. GerAA and GerAB are integral membrane proteins and GerAC is a putative lipoprotein. GerAA and GerAC, and GerBA, a GerAA homolog, are located in the inner membrane of the spore (Hudson et al., 2001
; Paidhungat and Setlow, 2001
) where they are positioned to detect germinants that can pass through the outer layers of the spore. The precise chemical nature of germinants varies according to the species, and although they are typically nutrients, these molecules are not metabolized. The amino acid L-alanine or a mixture of asparagine, glucose, fructose, and potassium ions germinates B. subtilis
spores, whereas L-proline germinates B. megaterium
spores and purine ribonucleosides and amino acids act as co-germinants for B. anthracis
spores (Setlow, 2003
High concentrations of nutrient germinants would be consistent with the ability of the environment to support the growth of germinated spores. However, a more integrated determination of this ability is the growth of other microbes in the environment, and this growth would be indicated by the presence of released muropeptides. How might dormant spores recognize these muropeptides? One protein sequence hypothesized to bind peptidoglycan is the PASTA (“p
hr kinase a
ssociated”) repeat found in the extracellular domain of membrane-associated Ser/Thr kinases as well as in some proteins that catalyze the transpeptidation reaction in cell wall synthesis. The PASTA domain is a small (~55 aa) globular fold consisting of 3 β sheets and an α helix, with a loop region of variable length between the first and second β strands (Yeats et al., 2002
). While the presence of PASTA domains in proteins that interact with peptidoglycan suggests that these domains may mediate this interaction, the binding of PASTA domains to peptidoglycan has not been demonstrated.
The cytoplasmic kinase domain of M. tuberculosis
PknB, the essential PASTA-domain-containing Ser/Thr kinase, is structurally homologous to eukaryotic Ser/Thr kinases (Young et al., 2003
). Consistent with this homology, PknB phosphorylates several proteins, including a transcriptional activator (Sharma et al., 2006
) and a cell division protein (Dasgupta et al., 2006
). The closely related B. subtilis
PASTA-domain-containing Ser/Thr kinase, PrkC, phosphorylates elongation factor G (EF-G) both in vivo and in vitro. EF-G is an essential ribosomal GTPase involved in mRNA and tRNA translocation (Gaidenko et al., 2002
), and although the activity of its eukaryotic homolog, eEF-2 (Ryazanov et al., 1988
), is regulated by phosphorylation, similar data are not available for EF-G. While PrkC is not essential, ΔprkC
strains have decreased viability (~1 log) following incubation in stationary phase for >24 hr (Gaidenko et al., 2002
) and are moderately defective for sporulation (Madec et al., 2002
Here we show that muropeptides, purified peptidoglycan or supernatants derived from cultures of growing cells, are potent germinants of dormant B. subtilis spores. Diverse bacteria can serve as the source of these molecules, but the identity of a single amino acid residue in the peptidoglycan stem peptide determines its ability to induce germination. PrkC is necessary for this germination response, and several small molecules known to affect the activity of related eukaryotic kinases either stimulate or inhibit germination.