For successful colonization, S. mutans
produces various mutacins to inhibit the growth of other competing species, thereby helping establish a flourishing biofilm in the oral cavity (27
). Mutacin-mediated cell killing is also considered a source of nutrients during nutrient-limited conditions (34
). This process is highly coordinated and regulated by the CSP-mediated ComDE quorum-sensing signaling pathway (23
). Petersen and colleagues (41
) recently isolated CSP-18, a truncated form of CSP (lacking the C-terminal three amino acids) that is functionally more potent than CSP-21, from the culture supernatant. However, the exact mechanism by which this truncated CSP-18 is generated in S. mutans
was unknown. Here, we set up a screening system to identify putative transcriptional regulators for mutacin gene expression in S. mutans
. Our screening yielded a cell surface-associated protease, SepM, which appears to cleave CSP-21 to generate the truncated CSP-18 peptide that acts as a true signaling molecule.
SepM encodes a Lon-like proteolytic domain at the C-terminal domain and a PDZ domain at the middle region. The Lon protease is a cytoplasmic serine protease consisting of hexameric rings of a single peptide chain carrying the peptidase domain, an AAA+ domain, and a domain with chaperone activity. Lon is essential for maintaining cellular homeostasis by mediating the degradation of nearly half of the abnormal or damaged proteins in E. coli
). Lon participates in numerous biological processes, such as quorum sensing, motility, biofilm formation, and stress responses (for a recent review, see reference 52
). Although Lon is widely distributed in various bacterial kingdoms, some members of the Firmicutes
, including streptococci, do not encode a true Lon protease. Whereas most of the Lon proteases characterized so far contain ATP binding motifs and reside in the cytosol, SepM does not possess any ATPase domain and is surface localized. Thus, this SepM protease is a unique class of Lon-like protease present in streptococci.
The active site of Lon is composed of a Ser-Lys dyad and shares no relation to the classical catalytic Ser-His-Asp triad of serine proteases (10
). SepM also contains a Ser-Lys dyad (S235 and K280), and when we mutated these two residues to Ala, the protein lost its catalytic activity to process CSP (data not shown), suggesting that SepM is truly a member of the Lon-like protease family. Consistent with this observation, we also found that treating cells with AEBSF inhibits the protein's ability to process CSP. Though it appears that SepM behaves like a Lon protease, several important fundamental differences do exist. For example, Lon degrades a variety of proteins, such as phage λ N protein and CcdA antitoxin, that are either denatured or naturally partially unfolded. Generally, Lon recognizes an N-terminal degradation tag that is rich in nonpolar amino acids (52
). Furthermore, Lon can degrade C-terminally SsrA-tagged proteins in E. coli
as well as in Mycoplasma
). Peptide bond hydrolysis is thought to occur in a processive linear manner from the N to the C terminus or vice versa. We have no evidence that suggests that SepM displays a processive protease activity; rather, SepM acts as a site-specific endoprotease and cleaves after the Ala residue at position 18 of the CSP. A recent report by Tian et al. (50
) suggests that the Ala18 residue is critical, since deletion or replacement of this residue completely abolished the signaling activity of the peptide. Thus, we speculate that SepM requires the Ala residue either for correct recognition or for optimum cleavage activity. Furthermore, SepM also needs all three residues after Ala for optimum activity, since deletion of the last Lys residue drastically reduces the peptide activity as well (49
Homologs of SepM have been found in a wide range of Gram-positive organisms, including all streptococci, bacilli, staphylococci, and lactobacilli. Sequence alignment suggests that the protein is highly conserved across the species (data not shown), yet the specificity of the CSP cleavage activity is highly restricted to S. mutans. For example, when synthetic CSP-21 was incubated with the cells from various streptococci, such as Streptococcus agalactiae, it failed to produce a positive signal, suggesting that CSP-21 is either not processed or improperly processed (data not shown). Thus, apart from the conserved Ser-Lys dyad (S235 and K280), other residues of SepM seem to play important roles in determining the substrate specificity. Further biochemical studies are required to fully understand the molecular mechanism of substrate recognition and specificity.
So far, CSP-21 is the only substrate that is recognized and cleaved by the SepM protease. However, it is very likely that SepM might be involved in the maturation of other peptide pheromones produced by S. mutans
. Mashburn-Warren et al. (32
) recently described a novel competence regulatory peptide (XIP) that is absolutely necessary for competence development. This XIP peptide is produced as a 17-residue propeptide that is processed outside the cell to generate the active seven-residue XIP pheromone (32
). Although SepM seems to be the ideal candidate for the XIP processing, SepM is probably not involved in XIP processing. If SepM were responsible for the maturation of XIP, then one would expect the ΔsepM
mutant strain to be completely competent deficient, which was not the case (). SepM might be involved in the inactivation of bacteriocins produced by other bacteria that are present in the dental plaque. In Streptococcus pyogenes
, Spy1356, the SepM homolog, has multiple functions. Spy1356 is crucial for surface expression of various proteins, including M protein, and responsible for binding with the extracellular matrix proteins (13
). Further studies are necessary to unravel the range of processes regulated by SepM and its homologs in other Gram-positive organisms.
The key question that remains to be answered is why S. mutans
employs an additional regulatory mechanism for the generation of the active signaling peptide when other streptococci do not use this strategy. There are several possibilities that may explain the need for the extra three amino acids at the C terminus. First, these terminal amino acids may provide protection against the proteolytic degradation that may occur in the dense multispecies biofilms. Indeed, CSP-21 is degraded by an S. gordonii
cell surface protease to provide a selective advantage to S. gordonii
in the natural habitat (53
). Second, the presence of the three amino acids may confer protection against its own surface proteases. Third, the intact CSP-21 may have additional functions other than participating in quorum signaling. For example, CSP-21 can act as a bacteriocin, and Petersen et al. (41
) have shown that CSP-21 can inhibit growth of other streptococci. Furthermore, Jarosz et al. (21
) have shown that CSP-21 inhibits the morphological switch from yeast to hypha formation in Candida albicans
The other important finding that emerged from our study was the identification of NlmTE as the transporter for CSP. S. mutans
encodes several ABC transporters, and the CslAB transporter was thought to be responsible for the CSP transport. This is because mutations in the cslAB
locus drastically reduce the natural transformation in S. mutans
). However, the data presented here support that NlmTE and not CslAB is the authentic transporter of CSP. For example, we found that the ΔnlmTE
mutant was unable to express PnlmA-gusA.
In addition, while the culture supernatant of the ΔnlmTE
mutant was not able to trigger the PnlmA-gusA
expression, the external addition of CSP was able to restore the PnlmA-gusA
expression in the ΔnlmTE
mutant (). Furthermore, the ΔnlmTE
mutant behaved the same way as the ΔcomC
mutant for natural transformation.
Fig 8 Model for the regulation of mutacin production in S. mutans UA159. Competence-stimulating peptide (CSP) is translated as a prepro-CSP (i). During secretion through the ABC transporter, NlmTE, the leader peptide, is cleaved to generate the 21-residue-long (more ...)
Based on our current results and existing evidence on mutacin production, we present a revised model for regulation of mutacin expression by the CSP peptide pheromone in S. mutans (). According to this model, CSP is translated as a 46-residue-long prepropeptide. This 46-residue peptide is transported through the NlmTE transporter, the same transporter that also secretes various mutacins. During transport, the leader sequence is cleaved off after the GG motif (present at position 25) by the proteolytic activity of NlmTE. The exported propeptide CSP further undergoes proteolytic cleavage at the C termini by the cell surface protease SepM. This matured 18-residue CSP peptide acts as a signaling molecule and turns on the ComDE two-component signal transduction pathway, which, in turn, positively regulates expression of various mutacin and mutacin-related genes. An important question that arises from this model is whether the processing of CSP by SepM is coupled with the secretion and whether the NlmTE transporter directly interacts with the SepM protease. Although we have no direct evidence, we believe that these two processes are not coupled. Experiments are under way to unravel the molecular details of CSP-mediated signaling in S. mutans and related streptococci.