Molecular Genetic Analysis of a Group AStreptococcusOperon Encoding Serum Opacity Factor and a Novel Fibronectin-Binding Protein, SfbX

Abstract

The group A Streptococcus (GAS) sof gene encodes the serum opacity factor protein, which is capable of opacifying mammalian sera and binding at least two host proteins, fibronectin and fibrinogen. The sof gene exists in approximately 50% of clinical isolates, and there is a classical association of so-called nephritogenic strains with the opacity factor-positive phenotype. In both a type emm49 strain and a type emm12 strain, the sequences upstream of the 5 end of sof and downstream of the putative terminator were determined to be nearly identical to a region in the M type 1 genome approximately 10 kb upstream of the emm1 gene. This close genetic linkage is likely reflected in the strict correlation of opacity factor phenotype with specific emm genotypes. A new fibronectin-binding protein gene, sfbX, was discovered immediately downstream of sof in emm12 and emm49 strains and in several other sof-positive strains. The sof and sfbX genes were found to be expressed on the same transcription unit, which was correlated with the putative promoter and rho-independant terminator sequences that flank these two genes. The sfbX genes from different emm types are predicted to encode ϳ650-residue surface-bound proteins sharing 89 to 92% sequence identity. SfbX residues approximately 1 to 480 are not highly similar to those of other known proteins, with the closest match being the Staphylococcus aureus coagulase protein. The remaining portions of these proteins (residues 481 to 650) contain four putative fibronectin-binding repeats highly similar to those of other streptococcal fibronectin-binding proteins and a potential LP(X)SG cell wall anchor motif. Targeted in-frame allelic-exchange mutagenesis, complementation, and heterologous-expression studies found that serum opacification is encoded by sof alone and that sfbX encodes a fibronectin-binding function. A recombinant SfbX protein was found to bind immobilized fibronectin and to partially inhibit GAS adherence to fibronectin. The sfbX gene was found to be present only in sof-positive strains, and together these genes could influence the spectrum of tissues colonized by sof-positive GAS.

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

“…The rmlD upR+erm and rmlD downF+erm primers were constructed with 30 bp 5′ extensions (underlined) corresponding to the 5′ and 3′ ends of the erm gene respectively. The upstream and downstream PCR fragments were combined with the 738 bp amplicon of the erm gene [amplified off the pDCerm plasmid (Jeng et al ., 2003)] as templates in a second round of PCR using primers rmlD upF and rmlD downR. The resultant PCR amplicon, containing an in frame substitution of rmlD with erm , was transformed into S. mutans  + p GacA and selected for Erm and Cm resistance as previously described (Perry et al ., 1983), to yield S. mutans Δ rmlD  + p GacA (SMUΔ rmlD  + p GacA ).…”

Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2

“…Because ftsY is the fourth orf in a putative operon, genomic DNA from mutant strain AH308 ⌬(orf1-orf2-orf3), which maintains the upstream promoter sequence, followed by the kan-resistance gene and ftsY structural gene, was used as the template. The PCR amplicon was ligated to the EcoRV site of the Streptococcus-E. coli shuttle vector pDCerm (37), and the resulting plasmid, (erm r and kan r ), was introduced into the ftsY null mutant, AH307, and NG8 by natural transformation. Transformants were selected on erythromycin.…”

The staphylocoagulase family of zymogen activator and adhesion proteins

“…Domain organization of ZAAPs. SC homologs are S. agalactiae proteins CAD46494 [34]; NP_687847 [33]; SfbX, Streptococcus pyogenes fibronectin (FN)-binding protein [37]; and vWbp [32]. The sequence that directs cell-wall sorting of SfbX is indicated by a diamond; the RGD triplet is also indicated.…”