Cloning and expression of the Staphylococcus aureus protein A gene in Escherichia coli.

A gene bank of Staphylococcus aureus strain Cowan I was established using an E. coli HB101/pBR327 host-vector system. Recombinants expressing staphylococcal protein A (SPA) were detected using an IgG-binding assay. A 3.2 Kb DNA fragment directing the synthesis of SPA in E. coli was identified. SPA produced by E. coli was characterised in minicells and by Western blotting and double diffusion experiments.     

Cloning and expression of the exfoliative toxin B gene from Staphylococcus aureus.

Exfoliative toxin type B is produced by bacteriophage group II strains of Staphylococcus aureus and is a causative agent of staphylococcal scalded-skin syndrome. In addition to exfoliative toxin B, most isolates also produce a bacteriocin and are immune to the action of the bacteriocin. These phenotypes, as well as resistance to cadmium, were lost after elimination of a 37.5-kilobase plasmid, pRW001, from S. aureus UT0007. Transduction and transformation showed that pRW001 carries the structural genes for four phenotypic characteristics of S. aureus UT0007: (i) exfoliative toxin B production, (ii) bacteriocin production, (iii) bacteriocin immunity, and (iv) resistance to Cd(NO3)2. The exfoliative toxin B structural gene (etb), which is located on a 1.7-kilobase HindIII fragment of pRW001, was cloned in the plasmid pDH5060 and transformed into phage group III S. aureus RN4220. Transformant clones produced extracellular exfoliative toxin B that was biologically active in the neonatal mouse assay. In the Escherichia coli genetic background, the exfoliative toxin B gene was expressed only after being cloned into the positive selection-expression vector pSCC31. The structural gene for cadmium resistance was also isolated on an HindIII fragment of pRW001 cloned in pDH5060. The loci for the exfoliative toxin B gene and the cadmium resistance gene(s) were identified on a restriction map of plasmid pRW001.     

Cloning and characterization of a gene for a 19kDa fibrinogen-binding protein from Staphylococcus aureus

Staphylococcus aureus has been shown to interact specifically with fibrinogen. Three different extracellular fibrinogen-binding proteins, two of which have coagulase activity, are produced by S. aureus strain Newman. The role of these fibrinogen-binding proteins during staphylococcal colonization and infection has not yet been fully elucidated. Here we describe the cloning, sequencing and expression of a gene for a 19kDa fibrinogen-binding protein. This gene, called fib, encodes a 165-amino-acid polypeptide, including a 29-amino-acid signal sequence. The recombinant protein, which has an estimated molecular mass of 15.9kDa, bound fibrinogen and was recognized by a polyclonal antiserum against the native Fib protein. Homologies between the Fib protein and the fibrinogen-binding domain of coagulase suggest that amino acids within this domain are involved in the binding to fibrinogen.     

Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

A gene of Staphylococcus aureus PS47 encoding lytic activity was cloned and expressed in Escherichia coli. Deletion analysis of a recombinant plasmid carrying a 7.4-kilobase-pair fragment (kbp) of S. aureus DNA suggested that the gene was located within a 2.5-kbp EcoRI-XbaI fragment. Analysis of extracts of E. coli harboring recombinant plasmids on denaturing polyacrylamide gels containing purified cell walls of S. aureus showed a clearing zone by a polypeptide of apparent Mr 23,000. The release of dinitrophenylalanine but not reducing groups from purified cell walls by a cell extract of recombinant E. coli suggested that we had cloned an N-acetylmuramyl-L-alanine amidase.     

Cloning and sequencing of sarA of Staphylococcus aureus, a gene required for the expression of agr.

To evaluate the effect of a sar mutation on the agr locus, Northern (RNA) blotting was performed to determine the levels of RNAIII, the agr regulatory molecule, in two isogenic pairs of Staphylococcus aureus strains. Our results demonstrated that RNAIII was either significantly diminished or absent in both sar mutants compared with the parents. The RNAIII level was partially restored in sar mutants complemented with an intact sar gene (designated sarA). Additionally, we were able to complement selected sar phenotypes with a plasmid carrying RNAIII (pRN6735). These studies suggest that the sarA gene is necessary for the optimal expression of agr. The sarA gene of strain RN450 was subsequently cloned and sequenced. Sequence analysis revealed an open reading frame of 372 bp with a predicted molecular size of 14,718 Da and a deduced pI of 8.52. The deduced protein sequence has a predominance of charged residues (33%) and shares sequence similarity with the virF gene of Shigella flexneri.     

金黄葡萄球菌fnbB基因的克隆及在大肠杆菌中的表达

金黄色葡萄球菌（Staphylococcus aureus）是引起奶牛乳房炎主要致病菌之一,主要通过其菌体表面的黏附素侵入寄主细胞引起疾病,为奶牛业造成巨大损失。金黄色葡萄球菌表面蛋白纤连蛋白结合蛋白(fibronectin-binding protein,FnBP)是其关键的黏附因子,在研制抗金黄色葡萄球菌的新型疫苗中占有重要地位.本文根据GenBank中纤连蛋白结合蛋白B基因（fnbB）序列设计特异性引物,以金黄葡萄球菌基因组DNA为模板,进行PCR扩增,获得3 458 bp 的DNA片段。使用T-A克隆技术,将PCR产物克隆至pGEM T easy Vector中,成功构建出了克隆质粒pGEM-fnbB。以 BamHI和XhoI 双酶切pGEM-fnbB和pET28a(+),并将纯化的基因fnbB 亚克隆至pET28a(+)中,构建出原核表达质粒pET28a-fnbB,并将其转化至E.coli BL21(DE3)感受态细胞中,经1 mmol/L的IPTG诱导和SDS-PAGE分析,在约165 ku 处出现了与预期目的蛋白相一致的外源蛋白带,Western blot分析结果表明该蛋白具有金黄葡萄球菌的抗原性。金黄葡萄球菌pET28a-fnbB成功表达为金黄葡萄球菌引起的奶牛乳房炎的诊断和研究新型疫苗奠定基础。     

Cloning of alpha-hemolysin gene from Staphylococcus aureus strain 015

The genomic library of Staphylococcus aureus O15 has been constructed on the EMBL-3 vector. The synthetic oligonucleotide probes to N- and C-end regions of alpha-hemolysin permitted identification of the recombinant bacteriophage clone RS-1 containing a gene for this protein. The restriction map of the cloned fragment has been constructed for restriction endonucleases SalGI, EcoRV, PstI, PvuII. Expression of the alpha-hemolysin gene in phagolysate of the recombinant clone RS-1 (1000 units per ml) has been demonstrated.     

Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli

Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.     

Cloning, sequencing, and expression of the DNA gyrase genes from Staphylococcus aureus.

We have isolated and cloned the gyrA and gyrB genes from Staphylococcus aureus. These adjacent genes encode the subunits of DNA gyrase. The nucleotide sequence of a 5.9-kb region which includes part of an upstream recF gene, the whole of gyrB and gyrA, and about 1 kb of unknown downstream sequence has been determined. The gyrB and gyrA gene sequences predict proteins of 886 and 644 amino acid residues, respectively, which have significant homologies with the gyrase subunits of Escherichia coli and Bacillus subtilis. Residues thought to be important to the structure and function of the subunits are conserved. These genes have been expressed separately by using a T7 promoter vector. N-terminal sequencing of the cloned gene products suggests that the mature GyrB subunit exists mainly with its initial five residues removed. Protein sequencing also supports the interpretation of our DNA sequencing data, which are inconsistent in several placed with the recently published sequence of the same genes (E. E. C. Margerrison, R. Hopewell, and L. M. Fisher, J. Bacteriol. 174:1596-1603, 1992).     

The N-terminal A domain of Staphylococcus aureus fibronectin-binding protein A binds to tropoelastin

Staphylococcus aureus is an important human pathogen. Its virulence factors include a variety of MSCRAMMs (microbial surface component recognizing adhesive matrix molecules), each capable of binding specifically to the host extracellular matrix. The fibronectin-binding protein, FnBPA, has been shown previously to bind immobilized fibronectin, fibrinogen, and alpha-elastin peptides. Here we show that region A of FnBPA (rAFnBPA) binds to recombinant human tropoelastin. Binding occurs to three separate truncates of tropoelastin, encompassing domains 2-18, 17-27, and 27-36, signifying that the interaction occurs at multiple sites. The greatest affinity was for the N-terminal truncate. We observed a pH dependency for the rAFnBPA-tropoelastin interaction with strong, nonsaturable binding at low pH. The interaction ceased at higher pH. These data support a model of surface-surface interactions between the negative charges present on rAFnBPA and the positive lysines of tropoelastin. A protein lacking the negatively charged C-terminal fibronectin-binding motif of the A domain of FnBPA and another construct lacking subdomain N1 were both capable of binding immobilized tropoelastin with a lower affinity. The binding properties of five site-directed mutants of rAFnBPA were compared with wild-type rAFnBPA. There was no decreased affinity for immobilized tropoelastin, in contrast to the defective binding of these mutants to alpha-elastin and fibrinogen. The data indicate novel interactions between tropoelastin and FnBPA that include the use of surface charges. These results demonstrate that FnBPA is capable of directly binding tropoelastin prior to its incorporation into elastin.     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.     

The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen

Staphylococcus aureus is an important pathogen capable of causing a wide spectrum of diseases in humans and animals. This bacterium expresses a variety of virulence factors that participate in the process of infection. These include MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that mediate the adherence of the bacteria to host extracellular matrix components, such as collagen, fibronectin (Fn), and fibrinogen (Fg). Two Fn-binding MSCRAMMs, FnbpA and FnbpB, have been previously identified. The Fn binding activity has been localized to the approximately 40-amino acid residue D repeats in the C-terminal part of these proteins. However, no biological activity has yet been attributed to the N-terminal A regions of these proteins. These regions exhibit substantial amino acid sequence identity to the A regions of other staphylococcal MSCRAMMs, including ClfA, ClfB, and SdrG (Fbe), all of which bind Fg. This raises the question of whether the Fn-binding MSCRAMMs can also bind specifically to Fg. In this report, we show that a recombinant form of the A region of FnbpA does specifically recognize Fg. We localize the binding site in Fg for recombinant FnbpA to the gamma-chain, in particular to the C-terminal residues of this polypeptide, the site also recognized by ClfA. In addition, we demonstrate that recombinant FnbpA can compete with ClfA for binding to both immobilized and soluble Fg. By the use of surface plasmon resonance spectroscopy and fluorescence polarization, we determine the dissociation equilibrium constant for the interaction of recombinant FnbpA with intact immobilized Fg and with a synthetic C-terminal gamma-chain peptide, respectively. Finally, by overexpressing FnbpA in a mutant strain of S. aureus that lacks the expression of both ClfA and ClfB, we show that native FnbpA can mediate the interaction of S. aureus with soluble Fg.     

金黄色葡萄球菌蛋白A (SpA) Z结构域串联体的克隆、表达和筛选

筛选一种高效重组金黄色葡萄球菌蛋白A(SpA)用于制备抗体纯化亲和介质。首先通过基因操作获得金黄色葡萄球菌蛋白A(SpA)的Z结构域单体、二串体、三串体、四串体和五串体基因,将目的基因分别克隆至pET-22b表达载体并转化至大肠杆菌BL21(DE3)感受态细胞,获得不同串联个数的Z结构域基因工程菌,经诱导表达和Ni2+亲和层析纯化得到Z结构域单体和二-五串体蛋白。纯化后的目的蛋白偶联至琼脂糖凝胶作为亲和层析介质,对人免疫球蛋白G(IgG)进行分离纯化。分析比较Z结构域串联体蛋白产量及其偶联的亲和介质对抗体吸附载量的差异。结果表明,构建的Z结构域单体、二串体、三串体、四串体和五串体基因工程菌能有效表达目的蛋白,制备的凝胶亲和介质可特异性吸附人IgG。增加Z结构域串联数,重组蛋白产量和单位摩尔数多聚体蛋白吸附载量获得提高,其中,重组四串体蛋白产量大(160 mg/10 g湿菌体),对抗体的吸附载量高(34.4 mg人IgG/mL胶),更适合作为配基用于亲和层析介质的制备。     

Cloning and expression of the Staphylococcus aureus glucosaminidase in Escherichia coli

Abstract A fragment of Staphylococcus aureus DNA encoding the glucosaminidase determinant was cloned in Escherichia coli by inserting the Sau 3A genomic fragments in the Bam HI site of the plasmid vector pBR322. One clone selected on the basis of its lytic activity was shown to contain a hybrid plasmid (pEU213) carrying a 4.7 kb insert of S. aureus DNA. Lytic activity was tested using different assays, and the enzyme production was confirmed by immunological reactions. An appreciable reduction of lytic activity was noted after few subcultures. The E. coli carrying pEU213 had a slower growth rate and increased autolytic activity compared to the parental strain. The possible reasons for this behavior are discussed.