Bacillus subtilis CwlP of the SP-β Prophage Has Two Novel Peptidoglycan Hydrolase Domains, Muramidase and Cross-linkage Digesting dd-Endopeptidase

Abstract

For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-β prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn2+-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn2+ dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined. For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-β prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn2+-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn2+ dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined. IntroductionThe cycle of bacteriophage infection of microorganisms comprises adsorption, insertion of nucleic acids, production of bacteriophage nucleic acids and proteins, and finally, host cell lysis. The infection cycle follows a highly ordered sequence of events where cell wall hydrolases, encoded in bacteriophage genomes, are involved in adsorption to cell walls (first infection cycle) and host cell lysis (final infection cycle) (1.Piuri M. Hatfull G.F. Mol. Microbiol. 2006; 62: 1569-1585Crossref PubMed Scopus (95) Google Scholar).One of the best studied Gram-positive bacteria, Bacillus subtilis, has prophage-like elements including PBSX, skin, and SP-β (2.Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessières P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3104) Google Scholar). The SP-β prophage is the largest prophage-like element in B. subtilis (2.Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessières P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3104) Google Scholar). Inspection of the BSORF database indicates that the SP-β prophage chromosomal region contains 184 genes and one cell wall hydrolase called BlyA, which has been identified as an l-alanine amidase (3.Regamey A. Karamata D. Microbiology. 1998; 144: 885-893Crossref PubMed Scopus (31) Google Scholar). Interestingly, the Pfam database predicts that another gene product, YomI (renamed CwlP (cell wall lytic enzyme related to phage)), has two cell wall hydrolase domains, a soluble lytic transglycosylase (SLT) 3The abbreviations used are: SLTsoluble lytic transglycosylaseRP-HPLCreverse phase-high performance liquid chromatographyDNPdinitrophenylA2pmdiaminopimelic acidGlcNAcN-acetylglucosamineMurNAcN-acetylmuramic acidMurNAcrMurNAc with a reduced endESI-MSelectrospray ionization-mass spectrometry. and peptidase M23 (Fig. 1).However, the function of the two domains (SLT and peptidase M23) remains unclear. BLAST searches of the BSORF and Pfam databases indicate that the SLT domain contains a soluble lytic transglycosylase and a muramidase, although the domain name is annotated “SLT.” At present, lytic transglycosylases and muramidases cannot be differentiated using amino acid sequence similarity. The peptidase M23 domain has hydrolytic activity, enabling it to digest the Gly-Gly bond. It is also known that LytM in Staphylococcus aureus has a peptidase M23 domain, which has been identified as a glycyl-glycine endopeptidase (4.Ramadurai L. Lockwood K.J. Nadakavukaren M.J. Jayaswal R.K. Microbiology. 1999; 145: 801-808Crossref PubMed Scopus (62) Google Scholar). However, B. subtilis LytH also has a peptidase M23 domain and has been shown to digest the l-Ala-d-Glu bond of spore peptidoglycans in vivo (5.Horsburgh G.J. Atrih A. Foster S.J. J. Bacteriol. 2003; 185: 3813-3820Crossref PubMed Scopus (26) Google Scholar). At present, the activity of the peptidase M23 domain of CwlP is unknown. The aim of this study, therefore, was to conduct a biochemical analysis of CwlP to enable us to predict its function.Here, we report that the SLT domain of CwlP exhibits hydrolase activity toward the B. subtilis cell wall and can digest the linkage between MurNAc and GlcNAc. The property is characteristic of a muramidase, not a lytic transglycosylase. We determined that the active site for hydrolysis is Glu1447. The M23 domain of CwlP was also found to exhibit hydrolase activity toward the B. subtilis cell wall as a dd-endopeptidase, which digests the d-Ala-diaminopimelic acid (A2pm) bond of the cross-linkage. The critical amino acid residues in the M23 domain comprise His1628 and His1660. Unusually, the d-alanyl-A2pm endopeptidase of the M23 domain of CwlP does not require Zn2+ ions, but Ca2+ for enzymatic activity.DISCUSSIONIn this study, we demonstrated that CwlP has muramidase activity but lacks lytic transglycosylase activity. The critical amino acid residue controlling the muramidase activity of the enzyme was found to be Glu1447 (Fig. 4B and supplemental Table S3). We previously reported that the N-terminal domain of CwlT exhibits muramidase activity, and that the critical amino acid residues comprised Glu87 and Asp94 (Fig. 4A) (19.Fukushima T. Kitajima T. Yamaguchi H. Ouyang Q. Furuhata K. Yamamoto H. Shida T. Sekiguchi J. J. Biol. Chem. 2008; 283: 11117-11125Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In that report, we hypothesized that Glu87 was the critical amino acid residue controlling hydrolytic activity, because other muramidases such as hen egg-white lysozyme (20.Malcolm B.A. Rosenberg S. Corey M.J. Allen J.S. de Baetselier A. Kirsch J.F. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 133-137Crossref PubMed Scopus (160) Google Scholar, 21.Matsumura I. Kirsch J.F. Biochemistry. 1996; 35: 1881-1889Crossref PubMed Scopus (54) Google Scholar), bacteriophage T4 lysozyme (22.Kuroki R. Weaver L.H. Matthews B.W. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8949-8954Crossref PubMed Scopus (81) Google Scholar), and goose egg-white lysozyme (23.Thunnissen A.M. Rozeboom H.J. Kalk K.H. Dijkstra B.W. Biochemistry. 1995; 34: 12729-12737Crossref PubMed Scopus (91) Google Scholar), have a conserved glutamic acid residue in the corresponding position that controls their catalytic activity. Because the glutamic acid residue is conserved in all muramidases characterized to date, including CwlP and lytic transglycosylases (such as Slt70), it appears likely that it could be involved in the hydrolytic activity of the enzyme. Nevertheless, Asp94 of CwlT also appears to be associated with hydrolytic activity, either directly or indirectly (19.Fukushima T. Kitajima T. Yamaguchi H. Ouyang Q. Furuhata K. Yamamoto H. Shida T. Sekiguchi J. J. Biol. Chem. 2008; 283: 11117-11125Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). However, Weaver et al. (24.Weaver L.H. Grütter M.G. Matthews B.W. J. Mol. Biol. 1995; 245: 54-68Crossref PubMed Scopus (124) Google Scholar) reported that goose egg-white lysozyme lacks a catalytic aspartic acid residue on the basis of their crystallographic analysis of the protein. Supporting this finding, inspection of the muramidase domain of CwlP shows that it has no candidate catalytic aspartic acid residue within its sequence (Fig. 4A). Therefore, it is possible that the aspartic acid residue found in a few characterized muramidases is not directly involved in hydrolysis of the glycosidic linkage of MurNAc-GlcNAc.The M23 domain of CwlP was able to digest linkage between d-Ala and A2pm, which comprises the cross-linked peptide side chains of the B. subtilis peptidoglycan (Fig. 5). It is known that the M23 family includes the Gly-Gly endopeptidases of the lysostaphin type (25.Kumar J.K. Appl. Microbiol. Biotechnol. 2008; 80: 555-561Crossref PubMed Scopus (111) Google Scholar) such as that found in S. aureus LytM and Staphylococcus capitis Ale1 (4.Ramadurai L. Lockwood K.J. Nadakavukaren M.J. Jayaswal R.K. Microbiology. 1999; 145: 801-808Crossref PubMed Scopus (62) Google Scholar, 26.Sugai M. Fujiwara T. Akiyama T. Ohara M. Komatsuzawa H. Inoue S. Suginaka H. J. Bacteriol. 1997; 179: 1193-1202Crossref PubMed Google Scholar). Very few enzymes within the metalloendopeptidase family (e.g. gp13 in the φ29 Bacillus bacteriophage), can digest d-Ala-A2pm linkages. However, no linkage digesting enzymes have been identified in “the M23 endopeptidase family,” as predicted from inspection of the Pfam database. Interestingly, CwlP-M23 exhibited no significant activity toward the S. aureus or S. thermophilus cell wall or peptidoglycan preparations (Fig. 7, C and D, and data not shown). Therefore, we conclude that CwlP-M23 cannot digest Gly-Gly and Ala-Ala linkages, and appears to specifically act as a d-alanyl-A2pm endopeptidase.We also found that the critical amino acid residues within CwlP-M23 were His1628 and His1660, because in our experiments, the mutated CwlP, H1628Q, and H1660Q proteins exhibited no hydrolytic activity (Fig. 6B and supplemental Table S3). The position of the conserved histidine residue (His1660) of CwlP is His291 in S. aureus LytM and His231 in S. capitis AleI; both of these positions have been identified as catalytic residues (27.Fujiwara T. Aoki S. Komatsuzawa H. Nishida T. Ohara M. Suginaka H. Sugai M. J. Bacteriol. 2005; 187: 480-487Crossref PubMed Scopus (11) Google Scholar, 28.Odintsov S.G. Sabala I. Marcyjaniak M. Bochtler M. J. Mol. Biol. 2004; 335: 775-785Crossref PubMed Scopus (88) Google Scholar). With regard to His1628 of CwlP, this conserved residue corresponds to His260 of LytM and His200 of AleI. Fujiwara et al. (27.Fujiwara T. Aoki S. Komatsuzawa H. Nishida T. Ohara M. Suginaka H. Sugai M. J. Bacteriol. 2005; 187: 480-487Crossref PubMed Scopus (11) Google Scholar) reported that His200 in AleI lies within one of the active sites, and Odintsov et al. (28.Odintsov S.G. Sabala I. Marcyjaniak M. Bochtler M. J. Mol. Biol. 2004; 335: 775-785Crossref PubMed Scopus (88) Google Scholar) indicated that His260 in LytM was a candidate catalytic amino acid residue. Hence, it appears likely that both histidine residues within CwlP-M23, LytM, and AleI are necessary for their endopeptidase activities.It is noteworthy that the other CwlP-M23 mutants, H1580Q, D1584N, and H1662Q, exhibited small amounts of hydrolytic activity (Fig. 6B and supplemental Table S3). The above three residues are also conserved in LytM and AleI where they appear to be metal binding sites (arrowheads in Fig. 6A) (27.Fujiwara T. Aoki S. Komatsuzawa H. Nishida T. Ohara M. Suginaka H. Sugai M. J. Bacteriol. 2005; 187: 480-487Crossref PubMed Scopus (11) Google Scholar, 29.Bochtler M. Odintsov S.G. Marcyjaniak M. Sabala I. Protein Sci. 2004; 13: 854-861Crossref PubMed Scopus (59) Google Scholar). Because CwlP-M23 needs divalent cations such as Ca2+, Co2+, and Mn2+ for its catalytic activity (Fig. 6C), it is predicted that His1580, Asp1584, and His1662 of CwlP are metal binding sites. Interestingly Zn2+ appears not to be an optimum divalent cation for CwlP-M23 activity (Fig. 6C), despite the fact that LytM and Ale1 are both Zn2+-dependent metalloendopeptidases (26.Sugai M. Fujiwara T. Akiyama T. Ohara M. Komatsuzawa H. Inoue S. Suginaka H. J. Bacteriol. 1997; 179: 1193-1202Crossref PubMed Google Scholar, 30.Firczuk M. Mucha A. Bochtler M. J. Mol. Biol. 2005; 354: 578-590Crossref PubMed Scopus (77) Google Scholar). φ29 Bacillus bacteriophage gp13 (d-alanyl-A2pm endopeptidase) also requires a Zn2+ ion for activity (31.Cohen D.N. Sham Y.Y. Haugstad G.D. Xiang Y. Rossmann M.G. Anderson D.L. Popham D.L. J. Mol. Biol. 2009; 387: 607-618Crossref PubMed Scopus (23) Google Scholar). We conclude, therefore, that the CwlP-M23 enzyme is unique because it does not depend on Zn2+ for catalytic activity.CwlP is a phage-related protein whose coding sequence is located in the SP-β prophage (2.Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessières P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3104) Google Scholar). At 2,285 amino acids in length, or 252 kDa, CwlP is the largest protein in the prophage region. Because CwlP has a phage-related minor tail domain, it is possible that it functions as a tail protein. Recently, Piuri and Hatfull (1.Piuri M. Hatfull G.F. Mol. Microbiol. 2006; 62: 1569-1585Crossref PubMed Scopus (95) Google Scholar) described the gp17 of the tape measure protein (Tmp), as a mycobacteriophage tail protein. The 1,229 amino acid Tmp protein contains a cell wall hydrolase that facilitates efficient infection of stationary phase cells (1.Piuri M. Hatfull G.F. Mol. Microbiol. 2006; 62: 1569-1585Crossref PubMed Scopus (95) Google Scholar). Interestingly, Kenny et al. (32.Kenny J.G. McGrath S. Fitzgerald G.F. van Sinderen D. J. Bacteriol. 2004; 186: 3480-3491Crossref PubMed Scopus (75) Google Scholar) showed that Orf50 of the bacteriophage Tuc2009 (906 amino acids) encodes a tail-associated cell wall-degrading activity that mediates infection through cell wall hydrolysis. However, it is not known whether CwlP functions as a tail protein. Further studies of CwlP function in infection with the point-mutated cwlP and null-mutated cwlP in vivo are ongoing in our laboratory.CwlP-SLT, CwlP-M23, and CwlP-SLTM23 showed no significant hydrolytic activity toward S. aureus and S. thermophilus cell walls (Fig. 7, C and D, and data not shown). Because the peptidase M23 domain of CwlP is a d-alanyl-A2pm peptidase, the enzyme should be able to digest target linkages accurately, resulting in no digestion of either cell wall or peptidoglycan. Although the muramidase domain of CwlP appears to be able to digest the glycan strands of various peptidoglycans, it was not able to digest S. aureus and S. thermophilus cell walls (Fig. 7, C and D, and data not shown). It is possible that modifications in these cell walls, such as the presence of anion polymers, may inhibit the hydrolytic activity of the muramidase domain of CwlP. In fact, our studies showed that the CwlP muramidase domain could digest B. subtilis, S. aureus, and S. thermophilus peptidoglycans, and E. coli cell walls, which have no anion polymers (Fig. 7, B–D, and supplemental Fig. S5). Consequently, the muramidase domain of CwlP appears to have a restricted hydrolase activity against cell walls.The full-length CwlP amino acid sequence is conserved in B. subtilis. S. aureus phage proteins (such as Q6GAK2_STAAS and Q8SDP3_9CAUD) are unique in having a three-domain structure, which includes a phage-related minor tail protein, a peptidase M23 domain, and a SLT domain. Their sizes are similar to the CwlP protein (>2,000 amino acids); however, outside the three domain regions, their sequences are quite dissimilar to CwlP. Accordingly, the SP-β phage (containing CwlP) can only infect B. subtilis cells, because digestion with CwlP-SLTM23 is restricted to B. subtilis cell walls.This is the first study of a newly categorized muramidase domain that has striking similarities with the E. coli Slt70 protein, both in the amino acid sequence and its catalytic residues. The fact that the peptidase M23 domain of CwlP is a unique Zn2+-independent d-alanyl-A2pm endopeptidase, means it was unlikely to have been included in the peptidase M23 family in the Pfam database. IntroductionThe cycle of bacteriophage infection of microorganisms comprises adsorption, insertion of nucleic acids, production of bacteriophage nucleic acids and proteins, and finally, host cell lysis. The infection cycle follows a highly ordered sequence of events where cell wall hydrolases, encoded in bacteriophage genomes, are involved in adsorption to cell walls (first infection cycle) and host cell lysis (final infection cycle) (1.Piuri M. Hatfull G.F. Mol. Microbiol. 2006; 62: 1569-1585Crossref PubMed Scopus (95) Google Scholar).One of the best studied Gram-positive bacteria, Bacillus subtilis, has prophage-like elements including PBSX, skin, and SP-β (2.Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessières P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3104) Google Scholar). The SP-β prophage is the largest prophage-like element in B. subtilis (2.Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessières P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3104) Google Scholar). Inspection of the BSORF database indicates that the SP-β prophage chromosomal region contains 184 genes and one cell wall hydrolase called BlyA, which has been identified as an l-alanine amidase (3.Regamey A. Karamata D. Microbiology. 1998; 144: 885-893Crossref PubMed Scopus (31) Google Scholar). Interestingly, the Pfam database predicts that another gene product, YomI (renamed CwlP (cell wall lytic enzyme related to phage)), has two cell wall hydrolase domains, a soluble lytic transglycosylase (SLT) 3The abbreviations used are: SLTsoluble lytic transglycosylaseRP-HPLCreverse phase-high performance liquid chromatographyDNPdinitrophenylA2pmdiaminopimelic acidGlcNAcN-acetylglucosamineMurNAcN-acetylmuramic acidMurNAcrMurNAc with a reduced endESI-MSelectrospray ionization-mass spectrometry. and peptidase M23 (Fig. 1).However, the function of the two domains (SLT and peptidase M23) remains unclear. BLAST searches of the BSORF and Pfam databases indicate that the SLT domain contains a soluble lytic transglycosylase and a muramidase, although the domain name is annotated “SLT.” At present, lytic transglycosylases and muramidases cannot be differentiated using amino acid sequence similarity. The peptidase M23 domain has hydrolytic activity, enabling it to digest the Gly-Gly bond. It is also known that LytM in Staphylococcus aureus has a peptidase M23 domain, which has been identified as a glycyl-glycine endopeptidase (4.Ramadurai L. Lockwood K.J. Nadakavukaren M.J. Jayaswal R.K. Microbiology. 1999; 145: 801-808Crossref PubMed Scopus (62) Google Scholar). However, B. subtilis LytH also has a peptidase M23 domain and has been shown to digest the l-Ala-d-Glu bond of spore peptidoglycans in vivo (5.Horsburgh G.J. Atrih A. Foster S.J. J. Bacteriol. 2003; 185: 3813-3820Crossref PubMed Scopus (26) Google Scholar). At present, the activity of the peptidase M23 domain of CwlP is unknown. The aim of this study, therefore, was to conduct a biochemical analysis of CwlP to enable us to predict its function.Here, we report that the SLT domain of CwlP exhibits hydrolase activity toward the B. subtilis cell wall and can digest the linkage between MurNAc and GlcNAc. The property is characteristic of a muramidase, not a lytic transglycosylase. We determined that the active site for hydrolysis is Glu1447. The M23 domain of CwlP was also found to exhibit hydrolase activity toward the B. subtilis cell wall as a dd-endopeptidase, which digests the d-Ala-diaminopimelic acid (A2pm) bond of the cross-linkage. The critical amino acid residues in the M23 domain comprise His1628 and His1660. Unusually, the d-alanyl-A2pm endopeptidase of the M23 domain of CwlP does not require Zn2+ ions, but Ca2+ for enzymatic activity.