Insertional mutagenesis of Bordetella pertussis adenylate cyclase.
We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.
Ladant
D
D
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Glaser
P
P
Ullmann
A
A
D000262:Adenylyl Cyclases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001886:Bordetella pertussis / Q000201:enzymology* / Q000235:genetics; D002147:Calmodulin / Q000378:metabolism; D002853:Chromatography, Liquid; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D015183:Restriction Mapping
D002147:Calmodulin; D003062:Codon; D000262:Adenylyl Cyclases