Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor.
An internal fragment of the cfb gene from group B streptococcal (GBS) strain R268 was amplified by polymerase chain reaction (PCR) using degenerate primers with sequences derived from the CAMP-factor amino acid (aa) sequence of GBS strain NCTC8181 [Rühlmann et al. (1988) FEBS Lett 235:262-266]. After cloning and sequencing this fragment, the remainder of cfb and the adjacent 5' and 3' sequences were amplified by inverted PCR of genomic DNA and directly sequenced from the PCR product. Within the 1560 bp sequenced, a complete cfb gene deviating in two deduced aa residues from the published sequence was identified. In addition, the cfbR268 sequence contained a 29-aa leader peptide. Using primers directed to the 5' and 3' ends of cfb for PCR, a cfb gene of uniform size could be detected in 19 clinical GBS isolates including three phenotypically CAMP-negative strains. Utilizing Northern blot analysis and primer extension assays, the cfbR268 promoter was located and the length of the cfb transcript was assessed at about 1100 bp. In a parallel experiment, no cfb transcript could be detected from the CAMP-negative GBS strain 74-360. The complete cfbR268 gene and different portions of its 5' and 3' ends were cloned into the plasmid pJLA602 and expressed in E. coli DH5 alpha. The recombinant peptides could be detected by Western immunoblots with polyclonal antiserum. Only the full-sized recombinant CAMP-factor was found to exert co-hemolytic activity in a sheep-blood agar assay. This co-hemolytic activity could be inhibited by anti-CAMP antiserum.
Podbielski
A
A
Institute of Medical Microbiology, Technical University (RWTH), Aachen, Germany.
Blankenstein
O
O
Lütticken
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics*; D006462:Hemolytic Plaque Technique; D008967:Molecular Biology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012756:Sheep; D013292:Streptococcus agalactiae / Q000235:genetics*
D001426:Bacterial Proteins; C039092:CAMP protein, Streptococcus; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D015345:Oligonucleotide Probes