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Molecular cloning, characterization, and complete nucleotide sequence of the gene for pneumolysin, the
Abstract:
A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.
Insights
Researchers isolated a recombinant bacteriophage expressing Streptococcus pneumoniae hemolysin, pneumolysin. This toxin
Area of Science:
- Molecular Biology
- Microbiology
- Bacteriology
Background:
- Streptococcus pneumoniae produces pneumolysin, a sulfhydryl-activated hemolysin.
- Pneumolysin contributes to pneumococcal pathogenesis.
Purpose of the Study:
- To clone and characterize the gene encoding pneumolysin.
- To analyze the expression and properties of recombinant pneumolysin.
Main Methods:
- Isolation of a recombinant lambda bacteriophage containing Streptococcus pneumoniae DNA.
- In vitro transcription-translation of the cloned gene in Escherichia coli.
- DNA sequencing of the pneumolysin gene and flanking regions.
- Biochemical characterization of hemolytic activity.
Main Results:
- A recombinant bacteriophage expressing active pneumolysin was obtained.
- Hemolytic activity was inhibited by cholesterol and neutralized by anti-streptolysin O serum.
- The pneumolysin gene encodes a 471-amino acid polypeptide with hydrophobic properties.
- Putative promoter and ribosome binding sites were identified upstream of the coding sequence.
Conclusions:
- The cloned gene successfully expresses active pneumolysin.
- The characterized pneumolysin shares properties with other sulfhydryl-activated toxins.
- The DNA sequence provides insights into pneumolysin's structure and regulation.