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Nucleic Acids Research, 1986, Vol. 14, No. 8 3251-3261
© 1986

Articles

Molecular cloning of pertussis toxin genes

Camille Locht, Paul A. Barstad1,2, John E. Coligan3, Leonard Mayer1,4, John J. Munoz, Susan G. Smith and Jerry M. Keith

Department of Health and Human Services, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratories of Pathobiology Bethesda, MD 20892, USA 1Microbial Structure and Function, Rocky Mountain Laboratories Hamilton, MT 59840 3Laboratory of Immunogenetics Bethesda, MD 20892, USA

Received January 28, 1986. Accepted March 11, 1986.

We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2–terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5–induced B. pertussis toxin–deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non–structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.

2Present address: Department of Veterinary Science, Louisiana State University, Baton Rouge, LA 70820

4Present address: Division of Bacteial Disease, Centers for Disease ControlAtlanta, GA 30333, USA


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