Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus.

The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.     

The gene sequence and some properties of protein H. A novel IgG-binding protein

The gene for protein H, a novel bacterial cell wall protein with specific affinity for human IgG Fc, was cloned from a group A Streptococcus and expressed in Escherichia coli. Recombinant E. coli cells produced two forms of a human IgG Fc-binding protein, one with an apparent Mr of 42 kDa in a periplasmic fraction and the other with an apparent Mr of 45 kDa in a mixed fraction of cytoplasms and membranes. Both 42-kDa and 45-kDa protein preparations similarly bound to human IgG1 to IgG4, human IgG Fc, and rabbit IgG, but not to IgG of mouse, rat, bovine, sheep, goat, and human IgA, IgD, IgE, and IgM. The complete nucleotide sequence of the cloned 1.8-kb DNA fragment was determined. An open reading frame encoded a hypothetical protein of 376 amino acid residues (Mr = 42,498). The N-terminal amino acid sequence, consisting of 41 residues, which was removed post-translationally had typical characteristics of Gram-positive bacterial signal peptides. Thus, the mature form of protein H was suggested to consist of 335 residues (Mr = 38,162). There were 3 repeated sequences consisting of 42 residues that were highly homologous to those of protein Arp, an IgA-binding streptococcal cell wall protein, and streptococcal M6 and M24 proteins. The C-terminal amino acid sequence consisting of 93 residues, directly following the repeated sequences, was also highly homologous to that of M6 and M24 proteins. No sequence homology was found between protein H and protein A or protein G, two other IgG-binding bacterial cell wall proteins.     

Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor.

Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previously isolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G. O. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encoding this plasmin receptor, plr, was isolated from a lambda gt11 library of chromosomal DNA from group A streptococcal strain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptor protein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli, resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of the gene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787. Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified. The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments of the purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded by plr. The deduced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54% identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.     

Synthesis and expression in Escherichia coli of the gene for the albumin-binding domain of Streptococcus protein G]

The chemical-enzymatic synthesis of a gene coding for A2B2 repeats of the albumin-binding domain of streptococcal protein G has been accomplished. The codon usage of the natural gene has been modified to adapt an artificial sequence for the efficient translation in E. coli. The gene (238 b.p.) was cloned in the polylinker plasmid pUCL1 and then fused in frame to the 3'-terminus of the gene for the IgG-binding domain of staphylococcal protein A, which was earlier cloned in the expression plasmid pUCL2. A fused polypeptide composed of the E and B domains of protein A and A2B2 repeats of protein G was produced in E. coli cells under the lac promoter control. The resulted product was isolated by affinity chromatography on IgG-sepharose and (or) albumin-sepharose.     

Analysis of protein A encoded by a mutated gene of Staphylococcus aureus Cowan I

The protein A (spa) genes from Staphylococcus aureus Cowan I and a mutant strain of Cowan I called V-1 earlier suggested to produce a monovalent IgG-binding protein A have been cloned in Escherichia coli. The DNA sequences coding for the IgG-binding part of the spa genes from both strains have been determined and compared with each other and with a partial amino acid sequence of purified protein A from strain V-1. The nucleotide sequence of the spa gene from strain V-1 reveals an NH2-terminally located IgG-binding region homologous to region E first reported for strain 8325-4, region D and the major portion of region A. The amino acid sequence analysis of the purified protein A from this strain also shows the presence of regions E and D but only a minor part of region A. Reversed-phase high-performance liquid chromatography fractionation of purified protein A from strain V-1 revealed that the preparation was heterogeneous, containing mainly two peptides with different abilities to bind IgG molecules. A shuttle vector containing the cloned protein A gene from V-1 was constructed and transformed into different strains of S. aureus and the produced protein A was purified and analysed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

Gene for an immunoglobulin-binding protein from a group G streptococcus.

The gene (spg) for an immunoglobulin G (IgG)-binding protein from a Streptococcus clinical isolate of Lancefield group G was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and 5'-flanking sequences was determined. The DNA sequence includes an open reading frame which encodes a hypothetical protein of 448 amino acid residues (Mr = 47,595). The 5' end of this open reading frame encodes a sequence resembling a typical secretion signal sequence, and the remainder of the encoded protein has features reminiscent of staphylococcal protein A and of streptococcal M6 protein, including repeated sequences and a similar C-terminal structure. Aside from this C-terminal structure, the encoded protein has little direct amino acid sequence homology to either protein A or M6 protein. In E. coli, the cloned gene directs the synthesis of a protein which binds to immunoglobulins, including rabbit immunoglobulin, goat IgG, and human IgG3(lambda). Its binding properties are similar to those of the protein G described by Bj?rck and Kronvall (L. Bj?rck and G. Kronvall, J. Immunol. 133:969-974, 1984), a type III Fc receptor from a group G streptococcus.     

Streptococcal protein G. Gene structure and protein binding properties

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.     

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.     

Extensive sequence homology between IgA receptor and M proteins in Streptococcus pyogenes

Many strains of Streptococcus pyogenes are known to express a receptor for IgA. The complete nucleotide sequence of the gene for such a receptor, protein Arp4, has been determined. The deduced amino acid sequence of 386 residues includes a signal sequence of 41 amino acids and a putative membrane anchor region, both of which are homologous to similar regions in other streptococcal surface proteins. The processed form of the IgA receptor has a length of 345 amino acids and a calculated molecular weight of 39544. The N-terminal sequence of the processed form is different from that previously found for a similar IgA receptor isolated from a S. pyogenes strain of type M60. The sequence of protein Arp4 shows extensive homology to the C-terminal half of streptococcal M proteins, but not to the streptococcal IgG receptor protein G or staphlyococcal protein A. Apart from the membrane anchor, this homology includes a sequence of 119 amino acid residues containing three repeated units and a 54-residue sequence without repeats. The protein expressed in Escherichia coli is found in the periplasmic space, in which it constitutes the major protein. Protein Arp4 is the first example of a surface protein that has both immunoglobulin-binding capacity and structural features characteristic of M proteins.     

Analysis and use of the serum albumin binding domains of streptococcal protein G

Streptococcal protein G is an IgG-binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG-binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG- and albumin-binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity chromatography, using IgG or HSA immobilized on Sepharose, showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin-binding. The structure of this part of the protein suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one-step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.     

Cloning and analysis of the gene (rpoDA) for the principal sigma factor of Pseudomonas aeruginosa

A gene (rpoDA) of Pseudomonas aeruginosa whose gene product has a homologous function and structure with the principal sigma factor of Escherichia coli was cloned and sequenced. The DNA region corresponding to one of the two hybridization signals found in P. aeruginosa DNA with a synthetic oligonucleotide probe (rpoD probe) was shown to be able to complement a temperature sensitive mutation of Escherichia coli rpoD gene. The amino acid sequence deduced from the nucleotide sequence of rpoDA showed an extensive homology with that of the principal sigma factor of E. coli throughout the entire region, which indicates that the two gene products have an essentially identical domain structure. A common basic structure observed among principal sigma factors of different eubacterial strains was proposed. RpoDA protein was identified in the extract of the cell carrying a plasmid clone with the rpoDA gene insert by Western blot analysis.     

Cloning and nucleotide sequence of a gene for Actinomyces naeslundii WVU45 type 2 fimbriae.

A genomic library of Actinomyces naeslundii WVU45 DNA in Escherichia coli was screened for antigen expression with rabbit antibody against A. naeslundii fimbriae. Western blotting (immunoblotting) of one recombinant clone carrying a 13.8-kilobase-pair insert revealed a 59-kilodalton (kDa) immunoreactive protein. A protein of similar electrophoretic mobility was detected from the isolated fimbrial antigen. Expression of the 59-kDa cloned protein in E. coli was directed by a promoter from the insert. The DNA sequence of the subunit gene was determined, and an open reading frame of 1,605 nucleotides was identified which was preceded by a putative ribosome-binding site and followed by two inverted repeats of 14 and 17 nucleotides, respectively. The reading frame encoded a protein of 534 amino acids (calculated molecular weight, 57,074), and the N-terminal sequence resembled that of a signal peptide. The presence of a 32-amino-acid signal peptide was indicated by amino-terminal sequencing of the fimbriae from A. naeslundii. The sequence, as determined by Edman degradation, was identical to that deduced from the DNA sequence beginning at predicted residue 33 of the latter sequence. Moreover, the amino acid composition of the predicted mature protein was similar to that of the isolated fimbriae from A. naeslundii. Thus, the cloned gene encodes a subunit of A. naeslundii fimbriae.     

Identification, isolation, and characterization of the structural gene encoding the delta' subunit of Escherichia coli DNA polymerase III holoenzyme.

The gene encoding the delta' subunit of DNA polymerase III holoenzyme, designated holB, was cloned by a strategy in which peptide sequence was used to derive a DNA hybridization probe. The gene maps to 24.95 centisomes of the chromosome. Sequencing of holB revealed a 1,002-bp open reading frame predicted to produce a 36,936-Da protein. The gene has a ribosome-binding site and promoter that are highly similar to the consensus sequences and is flanked by two potential open reading frames. Protein sequence analysis of delta' revealed a high degree of similarity to the dnaX gene products of Escherichia coli and Bacillus subtilis, including one stretch of 10 identical amino acid residues. A lesser degree of similarity to the gene 44 protein of bacteriophage T4 and the 40-kDa protein of the A1 complex (replication factor C) of HeLa cells was seen. The gene, when placed into a tac promoter-based expression plasmid, directed expression of two proteins of similar size. By immunodetection with anti-holoenzyme immunoglobulin G, both proteins are judged to be products of holB.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

A structurally novel staphylococcal protein A from the V8 strain

Protein A from the Staphylococcus aureus strain V8 has a molecular mass about 8000 Da less than that of known proteins A. The corresponding gene was cloned and expressed in Escherichia coli. Sequence analysis of this structurally new protein A revealed that it lacked an IgG-binding domain (58 amino acids), and that it also lacked two octapeptide repetitions located in the membrane/wall attaching region. Contrary to what had been proposed previously, the first translated amino acid is probably not a leucine, but very likely a methionine located 12 residues upstream.     

Solubilization of IgG-binding proteins from group A and G streptococci

The release of IgG-binding proteins from the cell surface of streptococcal strains AR-1 and G148 with various proteolytic enzymes, acid, alkali or SDS was investigated. The IgG-binding proteins were purified by affinity chromatography using IgG-Sepharose Fast Flow. After SDS-polyacrylamide gel electrophoresis and immuno-electroblotting the major proteins identified varied in relative molecular mass from 15,000 to 65,000 depending on the solubilizing agent used. The results showed that solubilization with trypsin gave the highest yield of IgG-binding proteins, that strain G148 yielded about twice the amount of protein as strain AR-1, and that elastase released an IgG-binding protein of high relative molecular mass of 65,000.     

Cloning, sequencing and expression of the sialidase gene from Actinomyces viscosus DSM 43798.

Chromosomal DNA from Actinomyces viscosus was digested with restriction endonucleases and the fragments ligated with pUC-vectors were used to transform Escherichia coli cells. Clones bearing the required sialidase gene were detected by spraying the colonies with the fluorogenic sialidase substrate MU-Neu5Ac. The identity of the cloned sialidase was confirmed after the 5700-fold enrichment and comparison with the purified enzyme of A. viscosus. Both sialidases were identical with regard to molecular mass, substrate specificity tested with sialyllactoses, and the inhibition of their activity by heterologous antisialidase antibodies. The sequenced insert (EMBL accession number X62276) revealed a mol% G + C of 68.2, typical for A. viscosus. An open reading frame of 2739 bp follows a sequence with dyad symmetry and an AG-rich region, and codes for 913 amino acids representing a molecular mass of 113 kDa. The conserved amino acid sequence [Ser-X-Asp-X-Gly-X-Thr-Trp] typical for bacterial sialidases was found at five positions in the predicted amino acid sequence. The gene of this enzyme is expressed by E. coli, despite the low relatedness of both species.     

A novel,anchorless streptococcal surface protein that binds to human immunoglobulins

We have characterized a novel surface protein from urea extract of whole cells of group A Streptococcus pyogenes (GAS). A major protein band (35kD) was found to hybridize with human IgG by Western blotting. A search of the N-terminal amino acid sequence of this protein by using the GAS genome sequence database revealed an open reading frame that encoded a 38-kDa protein with a signal peptide sequence. We have named this protein streptococcal immunoglobulin-binding protein 35 (Sib35). It was found to be an anchorless protein with no LPXTG motif, distinct from the M protein superfamily exhibiting immunoglobulin-binding activity, and partially secreted in the culture supernatant. Recombinant Sib35 was also shown to bind human IgA and IgM. The sib35 gene was found in all GAS strains examined, but not in oral, group B, C, or G streptococcal strains. These results suggest that Sib35 is a unique immunoglobulin-binding protein in GAS.     

Characterization of the sat 4 gene encoding a streptothricin acetyltransferase in Campylobacter coli BE/G4

Abstract The sat 4 streptothricin resistance gene from Campylobacter coli BE/G4 was cloned into pUC18, and its nucleotide sequence was determined. Streptothricin acetyltransferase activity was detected in Escherichia coli cells containing recombinant plasmid pAT132 which carries the sat4 gene as an insert. The deduced amino acid sequence displayed 21–27% amino acid identity with streptothricin acetyltransferases from E. coli and streptothricin producers Streptomyces lavendulae and Streptomyces noursei . The sat 4 gene was detected by hybridization in clinical and environmental isolates of Campylobacter spp.