Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis*

Background: Lactobacillus rhamnosus GG produces pili assembled and anchored by two distinct sortases, SrtC and SrtA, respectively. Results: Residue substitution within the triple glycine (TG) motif of pilin proteins impacts sortase-mediated polymerization. Conclusion: The TG motif determines sortase specificity during pilus biogenesis in GG. Significance: These results help explain the signaling involved in the assembly and anchoring of sortase-dependent pili. In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.

tic to pathogenic bacteria. However, the recent genome sequencing and analysis of Lactobacillus rhamnosus strain GG revealed the presence of a genomic island encoding three structural LPXTGlike pilin proteins called SpaC, SpaB, and SpaA and a pilin-specific sortase SrtC1 (4). L. rhamnosus GG 2 is globally marketed in probiotic products and has been among the best studied lactic acid bacteria (5)(6)(7)(8). In GG, the discovery that mucus-binding pili signal the intestinal cells has generated new insights into the mechanisms involved in host interactions and underlines the potential contribution of pili in probiotic response (9). However, it is not clear what the signaling mechanisms and molecular components are that are involved in this response.
Pili represent a major colonization factor in terms of host interaction, signaling, and also biofilm formation (10,11). Therefore, it is not surprising that such a phenotypic trait is found in a variety of bacteria colonizing mucosa-associated niches. Typically, the genes encoding the pilin subunits and its dedicated pilin-specific sortase(s) are organized in an operon-like cluster as observed in L. rhamnosus and other Gram-positive bacteria (4,12). The frequent presence of flanking transposable elements indicates that some bacteria may have acquired pilus gene clusters by horizontal gene transfer events (12). In L. rhamnosus GG, it was even demonstrated that the transposable elements located upstream from the pilus gene cluster constituted a functional promoter and contributed to its expression (13). In Gram-positive bacteria, sortase-dependent pilus structures result from the multimerization of major backbone pilins to which minor pilin subunits are incorporated, i.e. tip and stop pilins (2). In some species, it was shown that the tip and the stop pilins play roles in host interaction and/or pilus assembly (14,15).
The pilus biogenesis and anchoring relies on the central role of two functionally conserved sortases, i.e. pilus-specific sortase(s) and housekeeping sortase(s). The pilin subunits contain conserved domains that are required for secretion, anchoring, and pilus assembly (2). Typically, pilin precursors harbor a N-terminal peptide signal that allow secretion of the pilin subunits through the Sec-dependent secretion pathway (2). Following secretion and cleavage of its N-terminal peptide signal, the pilin subunit is retained to the cell membrane by its C-terminal hydrophobic domain (3). They are then covalently coupled to each other by isopeptide bonds that are catalyzed by pilin-specific sortases (12,14,16). Sortases specifically recognize the LPTXTG motif within the pilin subunits and cleave it between the threonine residue and the glycine residue (17). The resulting acyl-enzyme intermediate is a covalently bound pilinsortase complex. Next, the nucleophilic attack of the lysine residue of the pilin on the cysteine residue of the sortase results in oligomerization of pilin subunits (12,14,17). Through a similar mechanism, the pilus is elongated and then coupled to the housekeeping sortase. The thioester bond linking the basal pilin subunit of the pilus to the housekeeping sortase is attacked by the amino group of the precursor of peptidoglycan (lipid II). This results in the covalent coupling of the elongated pilus to the bacterial cell wall (17). It is assumed that the termination and attachment of the pilus is initiated once the housekeeping sortase is relaying the pilin-specific sortase. In Corynebacterium diphtheriae, it has been proposed that this sortase switch is dependent on a specific pilin subunit SpaB (18). However, different scenarios also exist; for instance, Bacillus cereus is lacking a stop pilin (19). One central question concerning the pilus completion and this sortase switch still subsists: what are the signals within the pilins that determine the sortase specificity (housekeeping versus pilin-specific sortases) and that allow a concerted and coordinated assembly of the different pilin subunits?
The aim of the present study is to further investigate these regulatory and signaling mechanisms in the pilus biogenesis of L. rhamnosus GG. Over the last few years, the details of the pilus biosynthesis in L. rhamnosus GG have been uncovered (9, 20 -23). Based on comparative analysis with other Gram-positive piliated bacteria, it became obvious that the structural organization and functionality of the pilus in L. rhamnosus GG is similar to a number of other bacterial species, such as Enterococcus faecalis and Enterococcus faecium (11,24). However, there are various differences that may explain their distinctive roles in the human intestinal tract. Using the gene sequences of all predicted LPXTG proteins in L. rhamnosus GG, we identified the conservation of some glycine residues juxtaposed to the canonical LPXTG motif. To examine the role of these residues within what is now called the triple glycine (TG) motif, we constructed a series of pili gene cassette that were recombinantly expressed and analyzed by transmission electron microscopy and immunoblotting. The present study aims at gaining insights into the pilus biogenesis and sortase specificity in L. rhamnosus GG and other piliated Gram-positive bacteria, where the presence of the TG motif potentially reflects similar regulatory mechanisms in pilus biosynthesis.
Construction of a Pili Cassette in L. lactis-Oligonucleotide primers were obtained from Oligomer Oy (Helsinki, Finland). Genomic DNA from L. rhamnosus GG was extracted using a Wizard genomic DNA purification kit (Promega), as previously described (4). Plasmids used in the present study are listed in Table 1. Phusion Hot Start II high fidelity DNA polymerase (Thermo Fischer Scientific), FastDigest restriction enzymes (Thermo Fischer Scientific), and T4 DNA ligase (Promega) were used as recommended by the manufacturers' instructions. The pili cassette pAC44 expresses the L. rhamnosus GG genes spaA and srtC1 under the control of the constitutive promoter P44 (47). The plasmid pAC44 was obtained as follows. The DNA fragment containing spaA and srtC1 genes was amplified by PCR and flanked with the restriction sites NcoI and SpeI. The DNA insert was restricted by NcoI and SpeI for 30 min at 37°C, and the digested insert was ligated to the similarly cut pNZ44 plasmid (47). The ligation mixture was then electroporated into L. lactis NZ9000, as previously described (48) and plated on GM17 agar plates with 5 g/ml chloramphenicol. Transformants were screened by colony PCR and then confirmed by restriction mapping and sequencing. Following a similar strategy, the plasmid pSpaA44 expressing the spaA gene was generated. Some of the plasmid constructs were also electroporated into other L. lactis strain derivatives (Table 1), as described above.
Directed Mutagenesis-Directed mutagenesis was performed to substitute amino acid residues located in the TG motif of the spaA gene. Each variant was generated using a similar approach. Briefly, the whole pAC44 plasmid was amplified by PCR using back to back phosphorylated primers, harboring the mutation(s) to introduce. Self-ligation, electroporation, and subsequent screening were performed as detailed above.
Cell Wall Extract Preparation and Immunoblotting Analysis-L. rhamnosus and L. lactis cell walls were isolated following the same procedure as previously described (49) with some changes. Bacterial cultures were adjusted to an A 600 nm of 1.0, washed with PBS, and disrupted by bead beating (3 ϫ 30 s) using sterile quartz beads (Merck). Cell lysates were resuspended in 500 l of PBS and pelleted by centrifugation (10,000 ϫ g) for 30 min. The cell wall-containing pellets were incubated for at least 3 h at 37°C in a 50-l solution containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 , 5 mM CaCl 2 , 10 mg/ml lysozyme (Sigma), and 150 units/ml mutanolysin (Sigma). Cell wall extracts were then ana-FIGURE 1. Identification of two distinct LPXTG signature motifs among the 19 predicted LPXTG proteins of L. rhamnosus strain GG. A shows the SG motif found in 15 LPXTG proteins, including the minor pilin subunits SpaB and SpaE, and the TG motif present in the major and tip pilin subunits, i.e. SpaA, SpaC, SpaF, and SpaD. Asterisks indicate the conserved glycine residues. B shows the two pilus gene operon identified in L. rhamnosus GG (4). Respectively, predicted tip pilins, major backbone pilins, minor pilins, and sortases are indicated by blue, light blue, magenta, and green arrows. The SG (magenta) or TG (blue) motif is also indicated for each gene, and residues of interest are underlined. lyzed by immunoblotting using rabbit SpaA antisera as primary antibody, as previously described (13).

Sequence Analysis of the C-terminal Domain of All Predicted
LPXTG Proteins in L. rhamnosus GG-The C-terminal regions of all 19 predicted LPXTG proteins in L. rhamnosus GG were retrieved and aligned (Fig. 1). Typically, these sequences showed a similar organization, i.e. a conserved LPXTG motif flanked by a positively charged hydrophobic domain. A closer look at the amino acid residues juxtaposing the canonical LPXTG motif revealed a high conservation of two glycine residues (positions ϩ6 and ϩ8; the residue numbering is based on the LPXTG motif, i.e. the leucine residue is on position ϩ1). Specifically, the conserved glycine residues (positions ϩ6 and ϩ8) are present in SpaA, SpaC, SpaD, and SpaF pilin subunits, whereas the putative stop transfer pilins (SpaB and SpaE) and the other predicted LPXTG proteins have a conserved glutamic acid or aspartic acid residue (position ϩ 6) (Fig. 1). The motif found in SpaA, SpaC, SpaD, and SpaF was termed triple glycine (TG) motif in contrast with the alternative motif called single glycine (SG) motif. SpaA and SpaC pilin subunits are assumed to be processed by the pilin-specific sortase SrtC1 during the elongation of the pilus structure in L. rhamnosus GG. SpaB putative stop pilin and other LPXTG proteins with the SG motif are predicted to be anchored to the peptidoglycan layer by the housekeeping sortase SrtA (21). We therefore hypothesized that secreted proteins with the TG motif may be preferentially recognized by pilin-specific sortases, allowing the multimerization of pilin subunits, whereas proteins with the SG motif would be recognized by the housekeeping sortase. Our observation parallels with the findings of Comfort and Clubb (50), who identified different LPXTG motifs associated with different sortase isoforms.
Sequence Analysis of the C-terminal Domain of the Pilin Subunits in Other Piliated Gram-positive Bacteria-Further analysis of pilin genes found in eight other Gram-positive species showed that the TG and SG motifs were consistently found . Asterisks indicate the conserved glycine residues. C illustrates a selected number of the pilus gene operons analyzed that were previously identified in earlier studies, as referred in the main text. The schematics are not representative of the overall orientation of pili clusters within their respective genome. Predicted tip pilin, major backbone pilin, minor pilin, and sortase are indicated by blue, light blue, magenta, and green arrows, respectively. Gray squares correspond to uncharacterized open reading frames located within pili gene clusters. The SG (magenta) or TG (blue) motif is also indicated for each gene, and residues of interest are underlined. MAY 30, 2014 • VOLUME 289 • NUMBER 22 within all pilin subunits, although the residues within the SG motif showed a slightly higher diversity than in the TG motif ( Fig. 2 and Table 2). Interestingly, as observed in L. rhamnosus GG, the TG motif was mostly found in predicted tip and major backbone pilins. The SG motif was associated with the predicted stop or base minor pilins. The genetic organization of the different pilus gene operons varies significantly, but the SG and TG motifs are mostly conserved, suggesting that similar signaling systems operate, possibly related to sortase recognition and specificity. In an effort to verify our hypothesis, mutagenesis analysis of the TG motif of the GG SpaA major backbone pilin was performed, providing a basis for comprehending the coordinated biosynthesis and assembly mechanism of pili in L. rhamnosus strain GG and other piliated Gram-positive bacteria.

Construction of a Pilus Gene Cassette in L. lactis-Construc-
tion of a pilus gene cassette in L. lactis has been previously used as a model to express or study sortase-dependent pili in L. rhamnosus and Streptococcus pyogenes (23,51). Using the same expression host, we constructed and cloned a minimal operon encoding the SpaA major backbone pilin and its dedicated pilinspecific sortase SrtC1. This operon was placed under the control of the constitutive promoter P44 and expressed in L. lactis subsp. cremoris strain NZ9000 (Figs. 3 and 4). Immunoblotting analysis and electron microscopy observations revealed that the operon was expressed at high level in L. lactis. The cells harbored long pilus structures distributed uniformly on the cell surface, whereas the wild type NZ9000 was pilus-less (Fig. 4). It is noteworthy that L. lactis NZ9000 encodes in its genome a housekeeping sortase SrtA. To exclude the possibility that L. lactis SrtA impacts on the SrtC1-mediated pilus polymerization reaction, the spaA gene only, without the srtC1 gene, was expressed in L. lactis NZ9000. This resulted in pilusless lactococcal cells, where SpaA proteins were retained as monomers on the cell surface (Figs. 3 and 4). Because this demonstrates the suitability of our expression system, it was used in conjunction with the minimal pilus operon spaA-srtC1 for subsequent mutagenesis and functional analysis.
Mutagenesis of the TG Motif and Impact on Pilus Polymerization-The LPXTG motif constitutes a key signal for sortase recognition, allowing the polymerization or anchoring of proteins to the bacterial cell surface. As mentioned above, the sortase motif clearly differs between SpaA/C subunits (LPHTG-GXG) and SpaB subunit (LPQTGDTV) (Fig. 1). In an effort to identify what amino acid residues are critical for SrtC1 recognition and pilus assembly, the residues at positions ϩ3, ϩ6, and

Sequences of the LPXTG motif of different pilin genes analyzed in the present study
The LPXTG motif is indicated in red. The residues constituting the SG and TG motifs are highlighted in green and blue, respectively. Sequences were recovered from sequence databases, as described in the main text. In the table, pili gene clusters from S. pneumoniae 19A-6 and S. agalactiae A909 are not shown because of their high similarity with clusters found in S. pneumoniae TIGR4 and S. pneumoniae 2603V/R, respectively. In some clusters, pilin genes are predicted to be nonfunctional, i.e. pseudogenes as shown for example in S. suis P1/7 by Takamatsu et al. (31). ϩ8 of the SpaA motif (LPHTGGXG) were replaced by the corresponding residues from the SpaB sortase recognition motif (Fig. 5). The replacement of the whole SpaA sortase recognition motif by the SpaB sortase recognition motif resulted in the lack of pilus structures in L. lactis. Only SpaA monomers or oligomers could be observed on the cell surface, indicating that the replacement of three residues (H3Q, G6D, and G8V) abolished the pilus formation. Subsequently, pilus operons with single substitutions at positions ϩ3, ϩ6, and ϩ8 were constructed to identify what amino acids at these positions were critical for pilus polymerization (Fig. 5). The H3Q substitution had no impact on the pilus assembly. This is consistent with previous studies on the LPXTG motif showing that the residue (X) between the proline and the threonine is interchangeable (52). The G8V substitution had a limited effect, i.e. the presence of slightly shorter pili (Fig. 5). Remarkably, the G6D mutation abolished the pilus assembly completely. No pili could be observed on the cell wall, indicating that the glycine residue on position ϩ6 is essential for pilus assembly and/or pilin-specific sortase recognition. In the L. rhamnosus GG genome, we observed that other predicted LPXTG proteins had a glutamic residue on that same position (ϩ6). These proteins are assumingly anchored to the peptidoglycan layer by the housekeeping sortase SrtA. Indeed, the G6E substitution within the SpaA protein sequence resulted in a similar phenotype as with the G6D mutant, i.e. lack of pili. For both G6E and G6D mutants, transmission electron microscopy showed some SpaA oligomers that could also be observed by immunoblotting analysis (Figs.  5-7). However, no long pilus structures were observed in G6E and G6D mutants. Expanding on this, we introduced the different constructs into L. lactis IL1403 srtA-null mutant (53) to investigate whether the lack of the housekeeping sortase SrtA impacts the pilus phenotype. Four different constructs were introduced into the lactococcal srtA-null mutant and analyzed by TEM (Fig. 8). They all resulted in formation of pili as in contrast with L. lactis NZ9000 harboring SrtA, indicating that the formation of SpaA pili is independent from the housekeeping sortase SrtA. However, it remains unclear how pili were retained onto the cell wall, possibly as SrtC1-pili intermediates. Detached pili were also observed by electron microscopy (data not shown). One construct with the G6D substitution was associated with a different phenotype. Whereas in L. lactis NZ9000, which produces SrtA, the G6D substitution was preventing pilus assembly, this was not the case in L. lactis IL1403 srtA-null mutant (Fig. 8). In the absence of SrtA, SpaA variants with the G6D substitution were processed by SrtC1 and assembled into pili.
Mutagenesis of the Glycine Residue ϩ6 of the TG Motif-To further delineate the contribution of the TG motif, we introduced into L. lactis NZ9000 a spaA-srtC1 operon where residue ϩ6 of the TG motif was substituted by all other possible amino acids. Subsequently, we characterized the resulting lactococcal strains by TEM and immunoblotting analysis (Figs. 6 and 7). As summarized in Table 3, in most cases, the replacement of the glycine residue (position ϩ6) resulted in the loss of pili, and only SpaA monomers or oligomers could be observed on the cell wall. The immunoblotting analysis revealed a series of distinct bands that may represent different SpaA n-mers, as proposed on Fig. 7. Wild type pilus structures were typically detected as a set of slow migrating bands with increasing sizes (ladder pattern), where most SpaA proteins were localized in  the higher molecular mass bands. Wild type-like pilus-producing phenotypes were observed only when the glycine residue of the TG motif was replaced by alanine and serine. The substitu-tion with basic residues lysine and arginine resulted in formation of some shorter and sparse pili (Fig. 6) that were detected by immunoblotting analysis only when overexposing the mem-   (coexpression of spaA-srtC1), where the residue (position ؉6) of the TG motif of SpaA has been replaced by all other amino acid residues. #, protein marker; X, mutant G6X, where X is the substituted amino acid; ', various monomeric forms of SpaA, i.e. full-length SpaA (35.9 kDa), peptide signal cleaved SpaA (ϳ32 kDa), and peptide signal and LPXTG cleaved SpaA (ϳ30 kDa); HMWL, high molecular weight ladder. Arrows indicate SpaA n-mers based on an estimation of SpaA n-mers molecular masses. SpaA n-mers associated to the cell wall may therefore correspond to a mass n ϫ ϳ30 kDa. Additional post-translational modifications may occur and were not included in the present calculations. Samples were grouped according to their pilus phenotype and/or the properties of the substituted amino acid X within the TG motif (position ϩ6).
brane (data not shown). It is noteworthy that in two cases, i.e. G6M and G6Q substitution, a low proportion of bacterial cells harbored few and short pili and were therefore not detectable by immunoblotting analysis. For other mutants analyzed, the pilosotype was homogeneous within the overall population and did show any pili formation, as summarized in Table 3.

DISCUSSION
The recent discovery of mucus-binding pili constituted a milestone in understanding how L. rhamnosus strain GG interacts with and signals its host (4,9,21,23). The genetic organization of L. rhamnosus GG pilus cluster is quite similar to some other piliated Gram-positive bacteria, such as E. faecalis (11). Therefore, a deeper comprehension of the mechanisms involved in the pilus biogenesis would not only benefit our general understanding of L. rhamnosus GG colonization and persistence in the gut but also provide insights into pilus assembly occurring in other Gram-positive bacteria.
In L. rhamnosus GG, all predicted LPXTG proteins possess either the TG or SG motifs (Fig. 1), allowing us to hypothesize on the role of these two conserved motifs in sortase specificity and recognition. This expands on the work by Comfort and Clubb that identified prevalent LPXTG motif for each sortase isoform (50). To verify our hypothesis, we established a L. lactis model encoding a minimal pilus cassette and a series of relevant mutated derivatives. The coexpression of SpaA and SrtC1 resulted in SpaA pilus structures on the cell wall of L. lactis, indicating that these two proteins are sufficient to generate the pilus backbone. The single production of SpaA pilins in the presence of SrtA did not yield to any pili, confirming that the SrtA function is restricted to protein anchoring only. In L. rhamnosus GG, SpaB and SpaC pilin subunits are also incorporated into the backbone (21) but most likely involved in other biological functions (4). The tip pilin SpaC has a TG motif and the putative stop pilin SpaB a SG motif, suggesting that SpaC might be similarly recognized by SrtC1 as SpaA.
In our L. lactis pilus model, the substitution of the glycine residues (position ϩ6) in the TG motif by acidic residues D or E (mimicking the SG motif) significantly altered pilus formation. Only SpaA oligomers were observed on the cell wall by TEM and immunoblotting analyses. Interestingly, in the absence of SrtA, the G6D SpaA pilins were polymerized by SrtC1. This showed that both G6D and G6E SpaA pilins may be preferentially recognized by the housekeeping SrtA but can still be processed by the pilin-specific SrtC1. This result may explain the lack of pili and the detection of G6D/G6E SpaA pilin aggregates/oligomers in the presence of both SrtA and SrtC1. In L. rhamnosus GG pilus biogenesis, SpaA and SpaC (TG motif) would be processed by SrtC1, whereas SpaB (SG motif) would be preferentially processed by SrtA, terminating the pilus polymerization upon incorporation of SpaB subunits into the pilus backbone (Fig. 9). The incorporation of SpaB within the backbone remains nevertheless possible, because both sortases appear to show certain substrate recognition flexibility. This is in agreement with previous observations where SpaB proteins were found in L. rhamnosus GG pilus backbone (21). Within the TG motif, the conservation of the three glycine residues clearly has an impact on the efficiency of the pilus polymerization reaction. In most cases, the substitution of the second glycine residues (position ϩ6) by other amino acid groups prevented the pili assembly. Only SpaA pilin subunits with an altered TG motif consisting of an Ala or Ser residue could result in the production of wild type-like pilus structures.
The TG/SG motif was not only found in L. rhamnosus GG but also in a number of other Gram-positive bacteria, such as C. diphtheriae, E. faecalis, S. pneumoniae, or B. cereus (Fig. 2 and Table 2). The genetic organization of the different pilus gene clusters greatly varies. However, we consistently detected the TG motif to be present in the predicted tip and major backbone pilin, whereas the SG motif was mostly found in the predicted stop/minor pilin subunit. In most cases, the TG motif is highly conserved among these tip and major backbone pilins, indicating that this TG motif may be required for the recognition of the tip and major pilin by the pilin-specific sortase, as shown in L. rhamnosus GG. It is noteworthy that the TG motif still allows some flexibility. As demonstrated, the replacement of the glycine residue (position ϩ6) by a serine did not impact on the pilus phenotype, which would explain why the SpaABC pili of C. diphtheriae NCTC 13129 is functional, although the SpaC pilin has the following TG motif: LPLTGSNG. Such a motif may be still recognized by the pilin-specific sortase. In contrast, the SG motif shows a higher diversity among species. Rather than being a truly conserved motif, the SG motif is a non-TG motif. The role of the TG motif in the concerted assembly of the pilin subunits is possibly based on the distinct affinity of the different sortase isoforms involved. As shown in the present study, one TG/SG motif is favored by one sortase isoform (SrtC/SrtA) but does not necessarily prevent its recognition by another. Our model may explain also the stoichiometry of the different pilins incorporated into the pilin backbone. In L. rhamnosus GG, the putative stop pilin SpaB has a SG motif, which is preferentially recognized by the housekeeping sortase SrtA. However, SpaB is found in a low proportion in the backbone (21), which may illustrate its weak but possible processing by the pilin-specific sortase SrtC1.
The remarkable conservation of the TG motifs in different pilus gene clusters from various Gram-positive bacterial spe-cies also suggests that similar regulatory pathways may intervene in terminating the pilus assembly by switching from pilinspecific sortases (polymerization) to housekeeping sortase (anchoring). In the present study, we illustrated the TG/SG motif through a number of 18 pilus gene clusters from nine species. However, it remains difficult to establish the extent of this motif across piliated Gram-positive species, and there are exceptions to this general pattern, as observed in some S. pyogenes strains for example. This does not necessarily exclude the possibility that a similar model occurs based on a distinct extended sortase recognition motif. The interaction between the pilin substrate, especially the TG/SG motif and the sortase, is functionally important but still remains unclear, because the sortase residues involved in substrate specificity have not been clearly identified. The identification of a flexible lid (DPF motif) located in the catalytic pocket of pilin-specific sortases was shown to be involved in regulating the catalytic activity of the enzyme (54 -56) but may not be involved in the substrate specificity. In the present study, the pilin-specific sortase SrtC1 of L. rhamnosus GG does not possess this conserved lid motif as observed in some other pilin-specific sortases. The remarkable conservation of the TG motif within pilin subunits indicates that other sortase residues possibly located in the vicinity of the catalytic site may control the substrate specificity. One cannot exclude the possibility that this specificity is due to the presence of the three glycine residues of the TG motif. Such motif would ensure greater chain flexibility nearby the LPXTG motif, promoting a better access to the sortase catalytic pocket. In contrast, the presence of the SG motif, which makes the chain more rigid, may be preferentially recognized by the housekeeping sortase. Elegant work by Suree et al. (57) using NMR spectroscopy data revealed the intimate interaction between For the G6K and G6R mutants, some short and sparse pili were observed by electron microscopy but could be detected by immunoblotting only when overexposing the blotting membrane, suggesting that only a low proportion of the bacterial population displayed short pili. ϩ, pili formation; Ϫ, no pili; ϩ/Ϫ, scarce pili.  the active site residues of S. aureus SrtA and the LPXTG motif and also proposed a mechanism of binding to lipid II, resulting in important data on the transpeptidation reaction. It is, however, not possible to predict the amino acid interactions outside the canonical LPXTG motif. Hence, a similar structural approach using the TG/SG motif would further contribute to understanding the docking details of sortases and their dedicated substrates in L. rhamnosus GG and other Gram-positive species. A recent report indicated that the structure of the SpaA pilin of L. rhamnosus GG is soon to be solved, bringing to light important data to support the present regulatory model (58).