Expression of the fibronectin-binding components of Streptococcus pyogenes in Escherichia coli demonstrates that they are proteins.
The fibronectin-binding components (fbcs) of two clinical isolates and a culture collection strain of Streptococcus pyogenes have been analysed. Western immunoblotting of bacterial lysates which had been fractionated on polyacrylamide gels revealed trypsin-sensitive fibronectin-binding species. The genes specifying the fbcs were cloned from all three strains and expressed in Escherichia coli using a lambda EMBL3 vector. An fbc gene from the culture collection strain was subcloned and expressed in the E. coli expression vector pJLA601, and subjected to deletion analysis. The fibronectin-binding domain was thereby localized within a 40 kDa truncated peptide encoded by the 1000 bp C-terminal region of the gene. Southern hybridization experiments demonstrated that the analysed gene was present in the parental S. pyogenes chromosome, but not in the DNA of fbc expressing lambda clones obtained from the two clinical isolates. Further evidence for the existence of at least two different types of fbcs in group A streptococci was provided by Western blot analysis of recombinant phage lysates which revealed a complex series of fibronectin-binding species ranging from 120 to 200 kDa in size and showing strain-dependent variation in their patterns. As was the case with parental streptococcal strains all of the recombinant fbcs were protease-sensitive, and treatment with trypsin or pronase resulted in a total loss of fibronectin-binding activity. Competitive inhibition experiments indicated that lipoteichoic acid was not a significant fbc in the tested streptococcal strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Talay
S R
SR
Department of Microbiology, Technical University of Braunschweig, Germany.
Ehrenfeld
E
E
Chhatwal
G S
GS
Timmis
K N
KN
D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D008070:Lipopolysaccharides; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000737:chemistry*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013297:Streptococcus pyogenes / Q000235:genetics* / Q000472:pathogenicity; D013682:Teichoic Acids / Q000494:pharmacology
D008070:Lipopolysaccharides; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D013682:Teichoic Acids; C009900:lipoteichoic acid