Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili*

Bacillus cereus and other Gram-positive bacteria elaborate pili via a sortase D-catalyzed transpeptidation mechanism from major and minor pilin precursor substrates. After cleavage of the LPXTG sorting signal of the major pilin, BcpA, sortase D forms an amide bond between the C-terminal threonine and the amino group of lysine within the YPKN motif of another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the BcpA sorting signal, resulting in a covalent bond between BcpA and the cell wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby positioning BcpB at the tip of pili. Thus, unique attributes of the sorting signals of minor pilins provide Gram-positive bacteria with a universal mechanism ordering assembly of pili.

Sortases catalyze transpeptidation reactions to assemble proteins in the envelope of Gram-positive bacteria (1). Secreted proteins require a C-terminal sorting signal for sortase recognition such that sortase cleaves the substrate at a short peptide motif and forms a thioester-linked intermediate to its active site cysteine (2)(3)(4). Nucleophilic attack by an amino group within the bacterial envelope resolves the thioester intermediate, generating an amide bond tethering surface proteins at their C terminus onto Gram-positive bacteria (5). Four classes of sortases can be distinguished on the basis of sequence homology and substrate recognition (6,7). Sortase A cleaves secreted protein at LPXTG sorting signals and recognizes the amino group of lipid II peptidoglycan precursors as a nucleophile (8,9). Sortase B cleaves protein substrates at NPQTN sorting signals (10). This enzyme immobilizes proteins within fully assembled cell walls, utilizing the cell wall cross-bridge as a nucleophile (11). Sortase C cuts LPNTA sorting signals and anchors proteins to the peptidoglycan cross-bridges in sporulating bacteria (12,13). Finally, sortase D catalyzes transpeptidation reactions in the assembly of pili (14,15). Sortase D recognizes the amino group of lysine residues within the YPKN motif of pilin subunits as nucleophiles (16). The resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow the pilus fiber (16,17).
Pili of Gram-positive bacteria comprised either two or three different pilin subunits synthesized as cytoplasmic precursors with N-terminal signal peptides and C-terminal sorting signals (P1 precursors) (14,18). After translocation across the plasma membrane, P2 precursor species arise from removal of the signal peptide from P1 precursors by a signal peptidase (16). Bacillus cereus pili are composed of two subunits; that is, the major pilin, BcpA, and the minor pilin, BcpB (15). In contrast to BcpA, which is deposited throughout the pilus, BcpB is found at fiber tip (15). Sortase D cleaves the BcpA LPXTG motif sorting signal between the threonine and glycine residues to form an amide bond to the ⑀-amino group of the lysine within the YPKN motif of adjacent BcpA subunits (16). However, sortase A also cleaves BcpA precursors, which are subsequently linked to the side chain amino group of meso-diaminopimelic acid within lipid II (19). The latter reaction serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall envelope (19).
The conservation of sortase D, the YPKN motif, and C-terminal sorting signal in major pilin subunits suggest a universal pilus assembly mechanism among Gram-positive bacteria (14,20). However, the molecular mechanism whereby bacilli deposit BcpB, the minor pilin, at the tip of BcpA pili is not known. Although the BcpB precursor harbors an N-terminal signal peptide and a C-terminal IPNTG sorting signal, it lacks the YPKN pilin motif of the major subunit (15). Furthermore, the substrate properties of the BcpB IPNTG sorting signal for the four classes of sortases expressed by bacilli has yet to be established.
Antisera-Polyclonal antisera to BcpA and BcpB were generated by injection of rabbits with purified His-tagged recombinant proteins (15).
Pilus Assembly-Bacilli were harvested after incubation for 20 h at 30°C in the presence of 1.5 mM isopropyl 1-thio-␤-Dgalactopyranoside and 20 M kanamycin. Bacilli were sedimented and boiled in 6 M urea, 1% SDS, 50 mM Tris-HCl, pH 9.5, for 10 min. The extracted material was washed with doubledistilled H 2 O, precipitated with 8% trichloroacetic acid, and washed once with 0.5 M Tris-HCl, pH 6.3, and then twice with 0.05 M Tris-HCl, pH 6.3. For immunoblots with ␣-BcpA and ␣-BcpB antisera, 3 ml of cell culture was digested with 100 units of mutanolysin in 0.05 M Tris-HCl, pH 6.3, 1.5 mM MgCl 2 , and 1 mM phenylmethanesulfonyl chloride. For purification of BcpB MH6 -BcpA from B. anthracis strains harboring pJB213, 6 liters of cell culture were digested with 20,000 units of mutanolysin, and cells were processed as described previously (19). Reactions were incubated at 37°C for 16 h, and the solubilized cell wall species was subjected to immunoblotting and purified by Ni-NTA affinity chromatography. For immunoblots of pili from B. anthracis Sterne variants (srtA::ermC), aliquots were precipitated with 7% trichloroacetic acid, incubated on ice for 30 min, and centrifuged at 16,000 ϫ g for 10 min. The sediment was washed with 500 l of acetone, air-dried, and suspended in 500 l of 4% SDS, 50 mM Tris-HCl, pH 8.0.
Purification of BcpB MH6 -BcpA-After cell wall digestion, insoluble material was removed by centrifugation at 33,000 ϫ g. The pH of the sample was adjusted to 7.5 with 2 M sodium phosphate dibasic, and BcpB MH6 -BcpA was purified by Ni-NTA affinity chromatography (11).
Purification of BcpB-BcpA Peptides-Purified BcpB MH6 -BcpA was methanol-chloroform-precipitated and cleaved with CNBr (11). BcpB H6 -BcpA was purified by a second round of Ni-NTA affinity chromatography under denaturing conditions and separated by reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection using a C18 column with a linear gradient from 1 to 99% acetonitrile (CH 3 CN) in 0.1% formic acid in 100 min, as previously reported for BcpA pilin peptides (16) and the BcpA anchor structure (19).
Mass Spectrometry-Aliquots of RP-HPLC fractions (0.5 l) were co-spotted with matrix (0.5 l of ␣-cyano-4-hydroxycinnamic acid) prepared at 10 mg/ml in CH 3 CN-water-trifluoroacetic acid (30:40:0.1). Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry spectra were obtained in a reflectron time-of-flight instrument (ABI Biosystems MALDI 4700) in reflectron mode. Spectra were acquired using external calibration with bovine insulin. Theoretical parent ion and fragmentation ion monoisotopic m/z values were produced with ProteinProspector version 5.1.8 Basic MSProduct webbased program (University of California, San Francisco).
Edman Degradation-RP-HPLC samples of anchor peptides were dried under vacuum and submitted for Edman sequencing at the University of Illinois, Urbana-Champaign Biotechnology Center Protein Sciences Facility. IsdX1-BcpB SS -GST cleavage NcoI AAAccatggTAATATACGATTTTTCTTATAGAAATA products were transferred to a polyvinylidene difluoride membrane, stained with Amido Black, and submitted for Edman degradation.

The Sorting Signal of the Minor Pilin Is Required for Its
Assembly into Pili-When transformed with pJB12, a plasmid encoding the B. cereus pilin operon (bcpA, srtD, bcpB), B. anthracis Sterne forms pili comprising both the major pilin subunit, BcpA, and the minor pilin subunit, BcpB ( Fig. 1A) (15). Lysates of bacilli were examined by SDS-PAGE and immunoblotting for the polymerization of pilin subunits. High molecular weight BcpA HMW and BcpB HMW species represent covalently linked pilin subunits within pilus fibers (Fig. 1B). As expected, B. anthracis expressing bcpA and srtD but not bcpB (pJB39) formed exclusively BcpA HMW species, indicating that the major pilin is polymerized in the absence of BcpB (Fig. 1B) (15). Substitution of lysine 162 of BcpA (K of the YPKN pilin motif) with alanine (pJB28, bcpA K162A , srtD, bcpB) abrogates polymerization of both BcpA K162A and BcpB into high molecular weight species (BcpB HMW ) and caused the accumulation of pilin precursors, BcpA P and BcpB P (Fig. 1B) (15). Deletion of the IPNTG peptide in the sorting signal of BcpB (pJB182, bcpA, srtD, bcpB ⌬IPNTG ) abolishes the formation of BcpB HMW without affecting BcpA polymerization (Fig. 1B). Thus, the BcpB C-terminal sorting signal is required for the incorporation of the minor pilin subunit into pili but is otherwise dispensable for the polymerization of BcpA.
The BcpB Sorting Signal Is Cleaved by Sortase D-To examine BcpB sorting signal cleavage, we generated a translational hybrid between the minor pilin 3Ј coding end and the 5Ј end of glutathione S-transferase (gst) (Fig. 2A). B. anthracis Sterne (wild-type sortase A) and an isogenic srtA deletion variant  (srtA::ermC) (21) were transformed with plasmids encoding the BcpB-GST hybrid, BcpA, and either wild-type sortase D (bcpA, srtD, bcpB-gst) or the active site mutant, C207A (bcpA, srtD C207A , bcpB-gst). Pilus assembly in bacilli was assessed by immunoblotting as before. Wild-type sortase D assembled BcpA HMW , whereas the C207A variant of sortase D did not form pili and accumulated the BcpA precursor (Fig. 2B). Similarly, wild-type sortase D yielded BcpB HMW , whereas the C207A sortase D variant accumulated BcpB-GST precursor (Fig. 2C). Immunoblotting with antibodies raised against purified GST detected the BcpB-GST precursor (P1/P2) as well as the mature C-terminal GST fragment released by sortase D cleavage (M, Fig. 2D). In contrast, bacilli expressing the C207A sortase D variant were unable to process minor subunit precursors. Furthermore, BcpB-GST cleavage is independent of sortase A, indicating that sortase D, but not sortases A, B, or C, is able to recognize BcpB substrate (Fig. 2D). Electron microscopy of immunogold labeled pili on the surface of bacilli demonstrates that GST fusion to the C terminus of BcpB does not affect pilus assembly (Fig. 2E). Mutants lacking sortase A successfully assemble pili on the bacterial surface, although they are abundantly shed into the extracellular medium (Fig. 2E). Mutants expressing the C207A variant of sortase D fail to form pili (Fig. 2E).
The BcpB Sorting Signal Determines Sortase D Substrate Specificity-B. anthracis as well as closely related B. cereus species secrete IsdX1, a hemophore that scavenges heme iron from host hemoglobin (22). We selected IsdX1 as a reporter because this protein is known to travel along the bacterial secretory pathway but is not associated with pili. IsdX1-BcpA SS -GST is a translational hybrid between IsdX1, the sorting signal of BcpA (BcpA SS ) and E. coli glutathione S-transferase (Fig. 3A) (19). Cleavage of the hybrid precursor (P1/P2) into the mature form (M) was monitored by immunoblotting with ␣-GST serum (Fig.  3A). IsdX1-BcpA SS -GST was cleaved in bacilli expressing sortase A and/or sortase D, indicating that the sorting signal of BcpA is recognized by both sortases (Fig. 3B) (19). The LPVTG peptide within BcpA SS was replaced with IPNTG, the peptide found in the BcpB sorting signal, thereby creating IsdX1-Hybrid SS -GST (Fig. 3A). Sortase A cleaves the IsdX1-Hybrid SS -GST less efficiently than the IsdX1-BcpA SS -GST. However, the abundance of mature products in bacilli expressing sortase D appeared similar for both substrates (Fig. 3B). A third fusion, IsdX1-BcpB SS -GST, encompasses the sorting signal of BcpB. This hybrid was cleaved in bacilli expressing sortase D but not in bacteria that lacked this transpeptidase (Fig. 3B). In bacilli expressing IsdX1-BcpA SS -GST, sortase A attached IsdX1 to the cell wall envelope. In contrast, sortase A-mediated cell wall sortase-cleaved products (M). B, sortase cleavage products were detected in urea-SDS-released cytoplasmic and membrane fractions by immunoblotting with ␣-GST antiserum. Antisera raised against B. cereus sortase D and B. anthracis sortase A allowed for their detection by immunoblotting. Labels indicate the sortase (srtA, srtD, or none) and substrate (isdX1 SS -gst with BcpA, BcpB, or hybrid cell wall-sorting signals) expressed in each strain. C, Bacillus cell wall extracts were digested with mutanolysin. IsdX1 anchoring was analyzed by immunoblot with ␣-IsdX1 antibodies. D, affinity chromatography of IsdX1-BcpB SS -GST from bacilli on glutathione-Sepharose revealed P1/P2 and mature (M) species, the latter of which was analyzed by Edman degradation. The experimentally determined amino acid sequences is printed in blue.
anchoring of IsdX1 could not be detected in bacilli expressing either IsdX1-Hybrid SS -GST or IsdX1-BcpB SS -GST (Fig. 3C). Lysates of B. anthracis Sterne expressing IsdX1-BcpB SS -GST were subjected to affinity chromatography on glutathione-Sepharose. Eluate was analyzed by Coomassie-stained SDS-PAGE, which revealed the P1/P2 precursors (50 kDa) and the mature sortase D-derived C-terminal cleavage fragment of IsdX1-BcpB SS -GST (31 kDa, Fig. 3D). The 38-kDa polypeptide in the eluate likely represents B. anthracis GST; this species did not react with antibodies against IsdX1 or E. coli GST (Fig. 3B). Edman degradation of the 31-kDa C-terminal sortase D cleavage product of IsdX1-BcpB SS -GST generated the amino acid sequence GGSGTTIFY, which matches the predicted sequence of the BcpB SS immediately after the threonine of the IPNTG motif (Table 2). Thus, sortase D cleaves the BcpB sorting signal between the threonine and the glycine of its IPNTG sorting signal.
Sortase D Incorporates BcpB into Pili of B. anthracis-We asked whether BcpB is incorporated into pili in the absence of sortase A, i.e. when these fibers cannot be immobilized in the cell wall envelope. B. anthracis (srtA::ermC) was transformed with pJB12 (bcpA, srtD, bcpB) or pJB39 (bcpA, srtD) (Fig. 4A). The srtA mutant bacilli released pili derived from either plasmid into the culture medium (Fig. 4C). Pili in the medium were precipitated with trichloroacetic acid and examined by immunoblot probed with ␣-BcpB and ␣-BcpA antisera (Fig. 4B). BcpA HMW was identified within polymerized pili derived from both plasmids, whereas BcpB HMW was only detected in the pili of pJB12 (bcpA, srtD, bcpB) transformants (Fig. 4B). The localization of BcpB in both cell-associated pili and released pili was established via immunogold labeling and electron microscopy. BcpA was detected with 10-nm immunogold conjugates along the shaft of pili that were displayed on the surface of bacilli or released into the culture medium (Fig. 4C). BcpB was detected with 15-nm immunogold conjugates at the tip of pili that were either displayed on the bacterial surface or released into the culture medium (Fig. 4C). BcpB-specific immunogold labeling was not detected in pili that were formed without bcpB (pJB39) (Fig. 4C). Together, these results suggest that sortase A is dispensable for the incorporation of BcpB into pili and that BcpB is deposited at the tip of BcpA pili irrespective of pilus surface attachment.
The Bond between BcpB and BcpA-B. anthracis (pJB48) encodes a mutant BcpA with a scrambled LPXTG motif sorting signal (LAVAA) that cannot be cleaved by sortases A and D and, consequently, cannot be incorporated into pili or the cell wall envelope (Fig. 5A) (15). As expected, immunoblotting of B. anthracis (pJB48) cell wall extracts did not reveal polymerized BcpA HMW or BcpB HMW (Fig. 5B) (15). Nevertheless, B. anthracis (pJB48) accumulated a 180-kDa species immunoreactive against both BcpA and BcpB sera (15). We reasoned these species must represent the sortase D transpeptidation product BcpB-BcpA LAVAA (15). If so, purification and biochemical analysis of this transpeptidation product may reveal the chemical bond that tethers BcpB to BcpA pili. To pursue this goal, we inserted an MH 6 tag two residues upstream of the IPNTG sorting signal of BcpB within plasmid pJB202 (bcpA LAVAA , srtD, bcpB MH6 ) (Fig. 5A). Asparagine 163 (YPKN 163 ) of BcpA forms an intramolecular isopeptide bond that is dispensable for pilus assembly, as substitution of asparagine 163 with alanine abolished isopeptide bond formation without affecting pilus assembly (16). As expected, introduction of the bcpA N163A mutation in pJB213 (bcpA N163A, LAVAA , srtD, bcpB MH6 ) had no effect on the ability of sortase D to generate the transpeptidation product BcpB MH6 -BcpA LAVAA (Fig.  5, A and B). As a control, the Hishorseradish peroxidase probe identified both BcpB MH6 precursor and FIGURE 4. SrtA is not required for the incorporation of BcpB into pilus fibers. A, plasmids expressing pilin genes under control of the P spac promoter were analyzed by immunoblot for pilus assembly in sortase A-mutant bacilli. pJB39 (39) expresses bcpA-srtD, whereas spJB12 (12) also expresses bcpB (see Fig. 1 for the schematic). Pili released into the medium were precipitated, separated by SDS-PAGE, and immunoblotted with ␣-BcpA (A) and ␣-BcpB (B). BcpA and BcpB high molecular weight material (HMW) and precursor species (arrowheads) are indicated. The electrophoretic mobility of the marker is indicated. C, pili released into the medium or attached to cells were labeled with ␣-BcpA serum and 10-nm gold anti-rabbit conjugate followed by ␣-BcpB serum and 15-nm gold conjugate. Arrowheads indicate 15-nm gold particles detected in pili from B. anthracis (srtA::ermC) (pJB12). Scale bars, 200 nm. Gly (12.14) 5 Thr (17.77) 6 Thr (17.21) 7 Ile (14.10) 8 Phe (11.06) 9 Tyr (10.45) BcpB MH6 -BcpA LAVAA in cell wall extracts from bacilli harboring pJB202 or pJB213 (Fig. 5B). Bacillus extracts containing BcpB MH6 -BcpA N163A, LAVAA were subjected to affinity chromatography on Ni-NTA-Sepharose (Fig. 6A). The eluate was analyzed by Coomassie-stained SDS-PAGE, which demonstrated purification of the BcpB MH6 -BcpA N163A, LAVAA transpeptidation product (Fig. 6A). Eluted transpeptidation product was cut at methionyl residues with cyanogen bromide (CNBr), and cleavage products were subjected to a second round of Ni-NTA affinity chromatography (Fig. 6A). Eluted peptides were further purified by RP-HPLC with UV detection at 215 nm (Fig. 6A). At 20% acetonitrile, 0.1% formic acid, a peak of 15-20 milliabsorbance units was detected, and the compound was subjected to MALDI-mass spectrometry (Fig. 6, A and B). The predominant ion signal at 2764.68 m/z was identified as the branched peptide HHHHH-HWVIPNT(YPKAEIKRGAM*) containing the tryptophan oxidation product hydroxytryptophan (calculated monoisotopic m/z 2764.41) (Fig. 6C). An additional cluster of peaks at 2782.69 m/z is thought to represent the branched peptide containing homoseryl instead of homoseryl lactone, methionyl reaction products that are acquired during CNBr cleavage (Fig. 6C) (16). The ion signal at 2764.68 m/z was fragmented via collisionally activated dissociation, and fragment ion spectra confirmed the predicted structure of the branched peptide displayed in Fig. 6D and Table 3). The b 5Ј and b 6Ј fragment ions revealed that the YPKN motif lysine residue 162 of BcpA participates in an amide bond with the C-terminal threonine (residue 717) of BcpB (Lys 162 -Thr 717 ) ( Table 3; Fig. 6D). The branched peptide was further analyzed by Edman degradation, which released A, schematic of plasmids expressing pilin genes under control of the P spac promoter. pJB28 expresses bcpA with the YPKN motif altered to YPAN (bcpA K162A -srtD-bcpB). pJB48 contains bcpA with an intact YPKN motif but harbors a scrambled sorting signal (bcpA LAVAA -srtD-bcpB). A MH 6 peptide was inserted two residues upstream from the IPNTG motif sorting signal of bcpB in pJB202, which also contained the scrambled sorting signal in bcpA (bcpA LAVAA -srtD-bcpB MH6 ). The YPKN motif in bcpA encoded by pJB202 was altered to YPKA, creating pJB213 (bcpA N163A LAVAA -srtD-bcpB MH6 ). B, cell wall extracts were digested with mutanolysin. Samples were separated by SDS-PAGE and immunoblotted with ␣-BcpA sera, ␣-BcpB sera, and His-horseradish peroxidase (HRP) probe. BcpB-BcpA LAVAA (BcpA-BcpB) and precursor species (P) are indicated. The electrophoretic mobility of the marker is indicated. FIGURE 6. An amide bond assembles BcpB and BcpA. A, BcpB MH6 -BcpA LAVAA was purified by Ni-NTA affinity chromatography from cell wall extracts treated with mutanolysin. Purified product was analyzed by SDS-PAGE and stained with Coomassie. The linked pilin proteins (BcpA-BcpB) and electrophoretic mobility of the marker are indicated. B, purified proteins were cleaved with CNBr, purified by a second round of Ni-NTA, and subjected to RP-HPLC with UV detection at 215 nm. mAU, milliabsorbance units. C, peptides that eluted at 20% acetonitrile, 0.1% formic acid were analyzed by MALDI-mass spectrometry. D, collisionally activated dissociation fragmentation spectrum of parent ion 2764.68 m/z. two amino acids per cleavage during the first two reaction cycles ( Fig. 6D; Table 4). In the third cycle, histidine was identified; however lysine (Lys 162 ) was not released during Edman degradation, as this residue is engaged in an amide bond with BcpB T 717 ( Fig. 6C; Table 4). In cycle 7 Edman degradation released the lysine of Lys 166 , which is not amidebonded to BcpA (Fig. 6D, Table 4). Tryptophan was not identified in cycle 7 because this residue is degraded during the Edman cycle (Table 4) (19). In summary, these results demonstrate that BcpB and BcpA are linked via an amide bond between the C-terminal threonine of BcpB and the ⑀-amino group of lysine within the YPKN motif of BcpA (Lys 162 -Thr 717 ) (Fig. 6D).

DISCUSSION
Many Gram-positive bacteria elaborate pili and thereby adhere to and invade host tissues or form biofilms (23). Minor pilin subunits are critically important for bacterial adherence to host cell surfaces, which is also a prerequisite for invasion or biofilm formation (24 -27). For example, the minor pilin subunit of Streptococcus agalactiae provides for bacterial adherence to the brain endothelium as well as pulmonary epithelial cells (28,29). Corynebacterium diphtheriae assemble pili from three subunits, the major pilin SpaA, and two minor pilins, SpaC and SpaB (14). SpaC is deposited at the tip of SpaA pili, whereas SpaB is found in regular intervals along the pilus shaft (14). Both SpaC and SpaB are required for corynebacterial adherence to pharyngeal cells, a process that does not depend on the SpaA subunit (30). Another minor pilin subunit, RrgA of Streptococcus pneumonia, promotes bacterial adherence to respiratory epithelia (31). RrgA has been reported to bind to the extracellular matrix components fibronectin, collagen I, and laminin (32). Finally, Group A streptococcal pili engage the scavenger receptor gp340 on pharyngeal cells to promote adherence and aggregation (33). Although minor pilins appear generally involved in pilus-mediated adherence, there are exceptions to this, as the major pilin subunit of S. pneumoniae pilus islet-2 (PI-2) mediates bacterial adherence to lung epithelial cells (34).
We have focused on B. cereus and its close relative B. anthracis to study the assembly of pili in Gram-positive bacteria. Bacilli form pili from two subunits, the major pilin BcpA and the minor pilin BcpB. We show here that the sorting signal of the minor pilin is recognized by sortase D, which subsequently cleaves its substrate between the threonine and the glycine residues of its IPNTG motif. The product of this reaction, a thioester-linked acyl enzyme, is resolved by the nucleophilic attack of the ⑀-amino group of lysine with the YPKN pilin motif of BcpA. The aforementioned reaction is not absolutely dependent on the LPVTG sorting signal of BcpA (15). We took advantage of this observation and purified the sortase D-catalyzed transpeptidation product BcpB MH6 -BcpA LAVAA using a BcpA sorting signal variant that cannot be used as substrate for further polymerization. Mass spectrometry and Edman degradation of affinity-purified BcpB MH6 -BcpA LAVAA revealed the amide bond that tethers the minor pilin subunit to the tip of BcpA pili.
The observation that the IPNTG sorting signal of BcpB is recognized by sortase D, but not by sortase A, provides a compelling argument for a model that may be universally applicable for pilus assembly in Gram-positive bacteria. Biochemical analysis of sortase-catalyzed transpeptidation reactions revealed that resolution of acyl intermediates is the rate-limiting activity of sortase (35). Thus, if BcpB were the preferred substrate of sortase D, its intermediates could only be resolved by the nucleophilic attack of the amino group of BcpA, generating BcpB-BcpA transpeptidation products and positioning BcpB at the tip of pili. Sortase D can only accept one nucleophile, the ⑀-amino group of lysine within the YPKN pilin motif of BcpA. As a consequence, sortase D cleavage of BcpB-BcpA must be followed by further polymerization with major pilin subunits, resulting in the formation of BcpB-BcpA nϩ1 . In contrast to BcpB, the BcpA LPVTG sorting signal can be cleaved by both sortase D and sortase A, the latter of which recognizes the side chain amino group of m-diaminopimelic acid within lipid II as a nucleophile to resolve its acyl intermediates (19). This reaction was recently demonstrated by studying the sortase A-catalyzed cell wall anchor structure of the major pilin subunit BcpA (19). Thus, competition between two transpeptidases, sortase D and sortase A, for the same pilin subunit can be viewed as a determinant for both pilus length and cell wall anchoring of fully assembled fibers. We propose that this model may be universally applicable to the assembly of pili in all Gram-positive bacteria. Pili in B. cereus, Actinomyces spp., and some group A streptococcal isolates are formed from two pilin subunits (36,37). However, other Gram-positive bacteria assemble pili from three subunits (38). C. diphtheriae pili are formed via polymerization of the major subunit, SpaA. One of the minor subunits, SpaC, is deposited at the tip, whereas another minor pilin, SpaB, is incorporated at regular intervals along the shaft of polymerized SpaA (14). Recent work suggests that SpaB encompasses a side chain amino group that functions as a nucleophile, producing reiterative covalent links between SpaA and SpaB subunits (39). Nevertheless, it is still not clear whether pilin-specific sortases recognize the side chain amino groups of both major and minor pilins as nucleophiles to resolve their intermediates. Furthermore, the amide bonds that are formed during the assembly of pili comprising three subunits must be revealed.