Pertussis toxin, a protein composed of five different subunits (S1, S2, S3, S4, and S5), is the major virulence factor of Bordetela pertussis. We have cloned and sequenced a DNA fragment of 4.7 kilobases that contains the genes coding for the 'five subunits. The genes are clustered within 3.2 kilobases in the following order: S1, S2, SA, S5, and S3. A sequence closely resembling Escherichia coli promoters is found only before the S1 gene, and a possible termination signal is present at the end of the S3 gene, which suggests that the pertussis toxin genes are organized in a single operon. A possible Shine-Dalgarno sequence is present before the S1 gene but not before the other four genes that 8-12 nucleotides upstream from the ATG codon show a new consensus sequence, 5'TCC(T)GG3', possibly involved in the regulation'of translation. We have also found sequence homology between the S2 and S3 genes and their protein products indicating that gene duplication played a major role in the evolution of pertussis toxin. (Fig. 2), they have a different mobility in NaDodSO4/PAGE. Note also that S5 is stained rather poorly and although its deduced molecular weight is smaller than that of S4 (Fig. 2) under reducing conditions, it migrates more slowly than S4.

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Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRMlOOl, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mabl 1.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM 197, CRM 176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.

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