Expression of M6 protein gene of Streptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptional fusion.
The M6 protein of Streptococcus pyogenes was expressed on the cell surface and secreted in Streptococcus gordonii Challis (formerly Streptococcus sanguis) after chromosomal integration of a promoterless M6 protein gene (emm-6.1). The ermC gene, conferring resistance to erythromycin, was cloned downstream of emm-6.1, within the same ClaI fragment. The initiation codon of emm-6.1 was 19 bp downstream of a ClaI site, so that ClaI cleavage would leave the gene promoterless. The ClaI fragment containing the promoterless emm-6.1 and ermC was ligated in vitro with a ClaI digest of S. gordonii chromosomal DNA. Random chromosomal integration of the heterologous DNA was obtained by using the ligation mixture to transform the naturally competent S. gordonii Challis. Twenty-eight percent of transformants selected for erythromycin resistance also expressed M6. Among the best M6 producers, 10 clones were selected for the stability of their phenotype. Nine of the 10 clones were shown to harbour one intact copy of the emm-6.1/ermC ClaI fragment integrated into the chromosome. These strains both expressed M6 protein on the surface and secreted different amounts of the molecule, since in each case the protein was produced after a transcriptional fusion of emm-6.1 with a different chromosomal promoter. A S. gordonii strain expressing large amounts of surface M6 protein, as judged by immunofluorescence and Western blot, was compared to the M- parental strain in a standard opsonophagocytosis assay. Of the isogenic pair, M6+ S. gordonii survived better in human blood and was phagocytosed at a slower rate.
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Dipartimento di Biologia Molecolare, Università di Siena, Italy.
Oggioni
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MR
Manganelli
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Fischetti
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D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D066298:In Vitro Techniques; D013297:Streptococcus pyogenes / Q000235:genetics*; D013298:Streptococcus sanguis / Q000235:genetics*; D014158:Transcription, Genetic; D014169:Transformation, Bacterial / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial