A protein G-related cell surface protein in Streptococcus zooepidemicus.

This work describes the cloning and sequencing of a gene encoding a plasma protein receptor from Streptococcus zooepidemicus. This receptor, termed protein ZAG, is a 45-kDa protein that binds alpha 2-macroglobulin (alpha 2M), serum albumin, and immunoglobulin G (IgG). The IgG-binding activity is located in the C-terminal part of the molecule and is mediated by two repeated domains highly homologous to each other as well as to the corresponding domains in streptococcal type III Fc receptors. The IgG-binding profile of protein ZAG is similar to that previously reported for S. zooepidemicus. Binding to serum albumin is mediated by a short amino acid sequence in the middle of the molecule. This domain shows homology to previously described albumin-binding proteins from streptococci, and the albumin-binding profile of protein ZAG is similar to that of streptococcal protein G. The N-terminal part of protein ZAG, which mediates binding to the plasma proteinase inhibitor alpha 2M, is composed of a unique stretch of amino acids. Protein ZAG competes for the same, or nearby, binding site(s) in alpha 2M as do two recently described Streptococcus dysgalactiae receptors, although the sequences of the alpha 2M-binding domains in these three receptors show only minor sequence similarities.     

Comparison of albumin receptors expressed on bovine and human group G streptococci.

The albumin receptor expressed by bovine group G streptococci was extracted and affinity purified. The protein was characterized for species reactivity, and monospecific antibodies were prepared to the purified receptor. The bovine group G albumin receptor was compared functionally, antigenically, and for DNA homology with the albumin-binding protein expressed by human group G streptococci. In agreement with previous reports, the albumin-binding activity of human strains was mediated by a unique domain of the type III immunoglobulin G-Fc-binding molecule, protein G. The albumin receptor expressed by bovine group G strains was found to lack any immunoglobulin G-binding potential but displayed a wider profile of species albumin reactivity than protein G. Both albumin receptors could inhibit the binding of the other to immobilized human serum albumin, and each displayed similar binding properties. Antigenic comparison of the two albumin receptors demonstrated a low level of cross-reactivity; however comparison at the DNA level, using an oligonucleotide probe specific for the albumin-binding region of protein G, demonstrated that the two albumin receptors expressed by human and bovine group G streptococcal strains do not display significant homology.     

Identification of a fibronectin-binding protein (GfbA) in pathogenic group G streptococci.

Attachment to eukaryotic cell surfaces is an essential step in the establishment of colonization and infection by bacterial pathogens. This report examines the adherence capabilities of pathogenic group G streptococci and demonstrates that certain group G streptococcal clinical isolates express a fibronectin-binding protein. This protein, termed GfbA for group G streptococcal fibronectin-binding protein, mediates adherence to human skin fibroblasts (HSF). The gene encoding this protein, gfbA, was isolated, and the complete DNA sequence of gfbA was determined. From this sequence GfbA was predicted to be a 580-amino-acid protein (molecular weight = 64,979) with significant amino acid identity to the group A streptococcal fibronectin-binding proteins SfbI and protein F (PrtF) (76 and 78% identity, respectively). GfbA contains regions with notable identity to the fibronectin-binding repeat domains of PrtF. gfbA(+) strains were able to bind to HSF, and preincubation of the gfbA(+) strains with fibronectin blocked this adherence. In addition, gfbA(+) strains were able to bind radiolabeled fibronectin, and this binding was inhibited with addition of excess unlabeled fibronectin. gfbA-negative strains were not able to bind either the HSF or radiolabeled fibronectin. DNA homologous to gfbA was found in 36% of the group G streptococcal isolates examined. Since not all group G streptococcal strains examined contained gfbA, this suggests there might be other tissue-specific adherence molecules expressed by these pathogenic strains.     

Binding of native alpha 2-macroglobulin to human group G streptococci.

Binding of human alpha 2-macroglobulin (alpha 2M) to group G streptococci and to their immunoglobulin G (IgG)-binding proteins (protein G) was investigated. Native alpha 2M bound specifically to strain G-148 with an apparent dissociation constant of (2.2 +/- 1.5) x 10(-9) M. Proteinase-complexed alpha 2M did not compete for the binding sites, and 125I-labelled proteinase-complexed alpha 2M did not bind to the bacteria. Binding of native alpha 2M to the cells was not affected by IgG or protein G consisting of only IgG-binding domains. 125I-labelled recombinant protein G did not bind to native or proteinase-complexed alpha 2M. However, a lysate of G-148 cells inhibited binding of alpha 2M to the bacteria, and immobilized wild-type protein G bound alpha 2M directly from fresh human plasma. In 13 group G streptococcal isolates, IgG-binding proteins were immunologically identified as protein G. In 11 isolates, these molecules reacted also with alpha 2M and human serum albumin (HSA). Western blots (immunoblots) of two wild-type protein G variants revealed identical bands reactive with goat IgG, HSA, and native alpha 2M. Digestion of wild-type protein G with clostripain destroyed in both variants the binding sites for alpha 2M but not for albumin and IgG. N-terminal fragments of protein G (lacking the IgG-binding region) bound both alpha 2M and HSA, whereas a similar HSA-binding peptide lacking the first 80 amino acids did not react with alpha 2M. Our findings are consistent with a specific binding site for native alpha 2M in the N-terminal region of protein G and suggest that binding of alpha 2M via IgG-binding proteins may be a general feature of human group G streptococci.     

Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgG

Protein G is expressed at the cell surface of certain group C and group G streptococcal strains. The protein shows a unique and specific affinity for the Fc region of mammalian polyclonal and monoclonal immunoglobulin G (IgG). We have cloned the streptococcal gene coding for protein G into E. coli, using phage lambda as the vector. The protein G produced by E. coli infected with this phage was detected and analysed in Western blot experiments using radiolabelled IgG Fc fragments as a probe. Three major IgG Fc-binding bands were obtained corresponding to apparent mol. wts of 47,000, 57,000 and 65,000, respectively. Analysis of the expression in E. coli indicates that this heterogeneity is caused by a post-translational degradation of the molecule before lysis of the lambda infected E. coli cells occurred. The protein G produced in E. coli was purified by affinity chromatography on IgG-Sepharose followed by gel-filtration on Sephadex G-200. This highly purified E. coli-produced protein G was compared to protein G solubilized by papain from streptococci, in direct binding experiments and in a competitive binding assay. The two protein G variants were found to interact with polyclonal IgG from different species in a similar way. Streptococcal strains expressing protein G also show affinity for human albumin, and at the molecular level protein G was found to be responsible also for the binding of albumin. Thus, both E. coli-produced protein G and the proteolytic fragment of protein G obtained from streptococci, bound albumin. On the protein G molecule, two different and separate sites were found to bind IgG and albumin. Finally, when whole streptococci were incubated with human plasma, the interactions with protein G caused a coating of the bacteria with albumin and IgG, whereas other plasma proteins showed no affinity for protein G.     

Binding of human fibronectin to group A, C, and G streptococci

A total of 387 bacterial strains belonging to 35 species were tested in direct binding experiments for the uptake of purified radiolabeled human fibronectin. Positive binding was found in group A, C, and G streptococci and in Staphylococcus aureus. The group C streptococcal species Streptococcus equisimilis, Streptococcus zooepidemicus, Streptococcus equi, and Streptococcus dysgalactiae were uniformly reactive with fibronectin. Beta-hemolytic bovine group G streptococci showed the same degree of reactivity as human group G strains. In contrast, only 4 out of 15 alpha-hemolytic bovine group G strains were able to bind fibronectin. The uptake of fibronectin measured at room temperature with a human group G streptococcus was a slow, time-dependent process with maximum binding after approximately 1 h. Human polyclonal immunoglobulin G and serum albumin tested in inhibition experiments did not affect the fibronectin binding. Fibronectin seems, therefore, to interact with a surface component that is different from the specific binding sites previously described for human immunoglobulin G and serum albumin.     

Effect of specific binding of human albumin, fibrinogen, and immunoglobulin G on surface characteristics of bacterial strains as revealed by partition experiments in polymer phase systems.

Four strains of gram-positive cocci with different combinations of positive binding of human proteins were investigated with respect to changes in physicochemical surface properties after specific protein binding. Staphylococcus aureus Cowan I, two group A beta-hemolytic streptococci, and one group G streptococcal strain were studied; they represented three different combinations of reactivity for human serum albumin, human immunoglobulin G, and fibrinogen. Using single-tube partition of bacterial cells in a dextran-polyethylene glycol system of constant polymer concentration but varying ionic compositions, it was possible to detect changes in the partition of bacteria after specific protein binding. There was a correlation between the binding of radiolabled human proteins to the bacterial strains and the effect of human proteins on the partition of the bacteria in the phase systems. Thus, the specific binding of proteins to the bacteria changes their physicochemical surface properties. These types of bacteria-protein interactions may play an important role in modulating host-parasite relationships.     

M12 protein from Streptococcus pyogenes is a receptor for immunoglobulin G3 and human albumin.

We previously showed that M12 protein from opacity factor-negative Streptococcus pyogenes (group A streptococci) CS24 is responsible for immunoglobulin G3 (IgG3) binding activity. Here, we report that this M protein binds human serum albumin (HSA). Deletion analysis showed that the C repeats are sufficient for binding HSA, although upstream regions may be required for optimal binding. Like protein G, IgG3 and HSA bind to independent domains in the M protein. Experiments showed that bound IgG3 did not inhibit HSA binding to the M protein. The interaction between M12 protein and HSA is specific. M12 protein does not bind chicken egg and bovine serum albumins. Alignments of C1 and C2 repeats of M12 protein to sequences at the carboxy termini of other M proteins and Ig receptors revealed highly homologous sequences in the FcRV, M5, M6, ML2.1, and M57 proteins, suggesting that all could bind HSA. As predicted from the alignment, M5 protein and M6+ streptococci bound HSA, whereas an isogenic M6- mutant did not bind HSA. Furthermore, M2 protein from an opacity factor-positive strain also bound HSA.     

Surface receptors for serum albumin in group C and G streptococci show three different types of albumin specificity.

A total of 100 bacterial strains were tested for binding uptake of radiolabeled albumin preparations from 15 mammalian species. Three types of surface structures with specific binding sites for albumin were defined. A previously described receptor for albumin was separated into type a in Streptococcus equisimilis strains and in human group G streptococcal strains and type b in bovine group C streptococci. A new type of albumin receptor, type c, was found in Streptococcus dysgalactiae strains, the only receptor type so far with high affinity for bovine serum albumin. Type of albumin receptor correlated with bacterial species. The three receptor types showed high binding capacities; 2 X 10(8) bacterial organisms bound from 5 to 16 micrograms of albumin. All types of albumin receptors were stable to heat treatment at 80 degrees C for 5 min, but susceptible to both pepsin and trypsin treatment. Bacteria-bound albumin preparations were eluted at various concentrations of KSCN, reflecting differences in affinity. Up to 500 micrograms of human fibrinogen or polyclonal human immunoglobulin G had no inhibitory effect on the uptake of albumin, indicating a separate molecular localization of receptors for these proteins.     

Binding of fibronectin to the surface of group A,C, and G streptococci isolated from human infections

Sixty-nine haemolytic and non-haemolytic streptococcal strains were isolated from various human infections and serogrouped with the coagglutination test. The amount of¹²⁵I-fibronectin bound to bacterial cells in a standard assay was determined. Most of the group A, C, and G strains were able to bind fibronectin. None of the group B or D strains bound significant amounts of fibronectin. Group A, C, and G streptococci appear to preferentially bind the N-terminal region of the fibronectin molecule because the 25K N-terminal fragment of the protein could effectively inhibit the binding of¹²⁵I-fibronectin to cells. Furthermore, the ability of representative strains of group A, C, and G to bind fibronectin was markedly reduced after trypsin treatment of the cells. Fibronectin binding components released from one strain by trypsin treatment inhibited the binding of¹²⁵I-fibronectin to all group A, C, and G streptococci strains. These findings indicate similarities among fibronectin binding proteins of the three groups of streptococci tested. However, the relative susceptibility to trypsin of fibronectin receptors of the three strains differed as did the relative potency of the inhibitory activity of receptors solubilized from different strains. Binding of fibronectin to the cell surface of group A, C, and G streptococci may contribute to virulence, for instance by promoting specific attachment to exposed fibronectin in open wounds and tissue lesions.     

Surface receptors for human serum albumin in Peptococcus magnus strains

Eighty-one bacterial strains representing 16 anaerobic species were tested in a sensitive binding assay for uptake of 125I-labelled human serum proteins. Fifteen of 36 Peptococcus magnus strains (42%) bound significant amounts of human serum albumin (HSA). None of the other bacterial species showed any affinity for HSA. All strains studied were incapable of uptake of human fibrinogen, fibronectin, haptoglobin or aggregated beta 2-microglobulin. P. magnus strain Ra 4 was tested for binding of purified serum albumin from 11 animal species, and showed a binding profile similar to human group-C and -G streptococci, but different from Streptococcus pyogenes, Strep. zooepidemicus and Strep. dysgalactiae. Kinetic experiments showed that albumin binding was a rapid displaceable, time-dependent process, that could take place over a wide range of pH or salt concentrations. The albumin-binding component of P. magnus strain Ra 4 was resistant to heat and to periodate treatment, but sensitive to proteolytic enzymes.     

Demonstration of specific binding sites for human serum albumin in group C and G streptococci.

A total of 297 bacterial strains belonging to 27 species was tested for quantitative uptake of radiolabeled human serum albumin. Specific binding sites with high affinity for human serum albumin were found exclusively in group C and G streptococci. The albumin binding was found to be a time-dependent, saturable, and displaceable process which obeyed simple kinetic equations. Scatchard analysis revealed that human serum albumin bound to a homogeneous population of receptors with an affinity in the order ot 10(7) liters/mol and that the average bacterial cell carried more than 80,000 binding sites. The albumin receptor is a heat-stable component susceptible to proteolytic digestion. It has a surface localization separate from the receptors for immunolgobulin G, fibrinogen, aggregated beta 2-microglobulin, and haptoglobin. In individual strains, albumin reactivity was also detected independently of these other types of interactions with human proteins.     

Extraction and characterization of IgG Fc receptors from group C and group G streptococci

Immunoglobulin G (IgG) Fc receptors (FcRs) were extracted by proteolytic digestion of four strains each of group C and group G streptococci. The solubilized proteins were analyzed in Western blots and multiple IgG-binding bands were obtained. The banding patterns of some of the strains were very similar, but this property was independent of which streptococcal group the strains belonged to. Highly purified FcRs were prepared from one group C and one group G strain. The 13 N-terminal amino acids were determined, and found to be identical, whereas comparison with the sequence of staphylococcal protein A did not reveal any homology. The isolated streptococcal FcRs also appeared closely related antigenically and functionally. Thus, both molecules were capable of inhibiting each others binding to immobilized IgG, and the radiolabelled group G FcR was completely inhibited from binding to IgG by an antibody to the group C FcR. Finally, in a direct binding assay both proteins were capable of reacting to a similar degree with a wide variety of IgGs, thereby demonstrating the great potential of streptococcal FcRs as tools for binding and detection of IgG antibodies.     

Interaction between streptococcal IgG Fc receptors and human and rabbit IgG domains.

Groups A, C and G streptococci were tested for their ability to bind 125I-labelled fragments of human and rabbit IgG in order to localize their sites of interaction with IgG domains. Among the Group A streptococci, strains with IgG Fc receptors bound 85% of the added IgG Fc fragments in the test systems, whereas these strains showed practically no reactivity with F(ab')2, Facb (F(ab')2 + C gamma 2 domains) or pFc' (C gamma 3 domains). The Group C and Group G strains bound 48-100% of IgG Fc, but could also bind up to 36% of the added F(ab')2 in accordance with a previously described 'alternative' Fab reactivity. However, unlabelled IgG F(ab')2 or Facb showed no, or only slight, inhibitory capacity for the binding of 125I-labelled IgG Fc to the C and G strains. Collectively, these results indicate that Groups A, C and G streptococci require both the C gamma 2 and C gamma 3 domains for interaction with IgG, and most probably also bind in the interface region between the C gamma 2 and C gamma 3 domains as has been shown for staphylococcal protein A.     

Identification and characterization of a novel fibronectin-binding protein on the surface of group A streptococci.

Understanding the role surface proteins play in the interaction of group A streptococci with epithelial cells is an important step toward the development of new strategies to fight infections. Fibronectin-binding proteins in streptococci and staphylococci have been described as important mediators for adherence to eukaryotic cells. In the present study we describe a new Streptococcus pyogenes fibronectin-binding protein (PFBP). The gene encoding the PFBP protein (pfbp) was identified from an M12 strain genomic library. It encodes a protein of 127.4 kDa which contains the LPXTGX motif characteristic of cell wall-associated proteins in gram-positive organisms and is among the largest surface molecules described for group A streptococci. The pfbp gene is transcribed during cell growth and was present in several class I and II streptococcal strains tested. The deduced amino acid sequence of PFBP exhibits a variable N-terminal region and a conserved C-terminal region when compared to most fibronectin-binding proteins identified from other gram-positive bacteria. The N-terminal region presents a stretch of 105 amino acids with no homology with N-terminal regions of previously described fibronectin-binding molecules, while the C-terminal region contains three repeat domains that share significant similarity with the repeat regions of fibronectin-binding proteins from S. pyogenes, S. dysgalactiae, and S. equisimilis. The PFBP repeated region, when expressed on the surface of S. gordonii, a commensal organism, binds to soluble and immobilized fibronectin. This study also shows that, in addition to pfbp, a second gene homologous with that of protein F1 (which also codes for a fibronectin-binding protein) is transcribed during cell growth in the same S. pyogenes strain.     

Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to epithelial cells.     

Receptor in group C and G streptococci detects albumin structures present in mammalian species.

The presence of albumin structures with the capacity to bind to a surface receptor in group C and G streptococci was studied in serum samples from 45 mammalian species representing 15 different orders, using an inhibition assay. The ability of animal sera to inhibit the uptake of radiolabeled human serum albumin by the streptococci indicated the presence of such albumin structures. Positive reactions were found in species of most orders tested, with Marsupialia as a notable exception. All Carnivora sera tested were strongly positive. In some orders such as Artiodactyla both positive and negative species were identified. Serum samples from 62 bird species representing 15 orders and from 5 fish species were also tested in the inhibition assay. None of these serum samples was capable of inhibiting the uptake of human serum albumin by streptococci. Some differences were also noted in the results obtained with group C and G streptococci from human and bovine sources, respectively, indicating the presence of two types of receptors. The present studies suggest a phylogenetic origin of albumin structures with affinity for the streptococcal receptor to a period after the divergence of Marsupialia from the other mammalian orders.     

Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.     

Molecular characterization of an IgA receptor from group B streptococci: sequence of the gene, identification of a proline-rich region with unique structure and isolation of N-terminal fragments with IgA-binding capacity

Certain strains of group B streptococci express a cell surface protein that binds IgA and acts as a virulence factor. This IgA receptor is referred to here as protein Bac. The gene for protein Bac was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. The deduced amino acid sequence of 1134 residues includes a signal sequence of 37 amino acids and a putative membrane anchor region at the C-terminal end. The processed form of the receptor, 1097 residues, has a calculated molecular weight of 123,786. There are no cysteines in protein Bac, suggesting a fibrillar structure. The C-terminal half of the protein includes a 90 residues long region with a novel type of periodic structure, the "XPZ motif", in which every third amino acid is proline. Unlike other bacterial immunoglobulin-binding proteins, there are no long repeats in protein Bac. Clones which express only part of the protein Bac gene were used to show that IgA-binding takes place in the N-terminal part of the molecule. Protein Bac was originally described as an antigen called beta, but N-terminal fragments that bind IgA do not react with a reference serum against the beta antigen. These and other data indicate that protein Bac can be divided into two regions with different functions: an N-terminal IgA-binding region and a C-terminal region corresponding to the beta antigen. The IgA-binding region of protein Bac does not show any homology to protein Arp, the IgA receptor from group A streptococci, although these receptors have similar binding properties. This indicates that convergent evolution has favored the appearance of these two structurally different streptococcal IgA receptors.     

Genomic location and variation of the gene for CRS, a complement binding protein in the M57 strains of Streptococcus pyogenes

All isolates of serotype M1 of group A streptococci possess a gene for streptococcal inhibitor of complement (SIC) in the mga regulon, which harbors genes for other virulence factors, such as M and M-like proteins, C5a peptidase, and a regulator. In serotype M57 the gene for a protein that is closely related to SIC (crs57) is located outside the mga regulon. We mapped the location of the crs57 gene in six strains of emm57 (gene encoding the M57 protein) sequence types to an intergenic region between the ABC transporter gene (SPy0778) and the gene for a small ribosomal protein (rpsU). The noncoding sequences on both sides of crs57 exhibited high degrees of identity to the corresponding regions of sic from M1 strains. This included one of the inverted repeat sequences of IS1562 but not the insertion element itself. These observations suggest that crs57 was recently acquired by serotype M57 or its progenitor via horizontal acquisition from serotype M1. The six emm57 sequence type isolates analyzed in this study belong to two distinct molecular types (vir types VT8 and VT101). Although the crs57 sequences from VT8 strains had very few substitution mutations, the VT101 crs57 sequence had a large number of such mutations. The CRS57 proteins from these strains are secretory products and have the ability to bind to complement proteins. All these proteins contain several tryptophan-rich repeats designated DWS motifs and internal repeat sequences. In all of these structural and biochemical characteristics CRS57 resembles SIC from M1 strains. Hence, CRS57 has a functional role similar to that of SIC in an M1 strain.