Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication.

Abstract

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.

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…Whereas T4SS-mediated secretion across the bacterial membranes is thought to normally function in a single step, the substrate of the Ptl system, the heterooligomeric pertussis toxin (PT), is secreted in two steps. First, the toxin subunits are exported into the periplasmic space, the export being carried out by the Sec system (Locht and Keith, 1986;Nicosia et al, 1986). Next, the subunits assemble into the holotoxin, which then becomes secreted to the exocellular milieu by means of the Ptl type IV secretion machinery (Weiss et al, 1993).…”

Identification of an active‐site residue in subunit S1 of pertussis toxin by photocrosslinking to NAD

“…Recent achievements in sequencing the PT gene [2,3] have stimulated interest in detoxifying the toxin by genetic engineering [4]. PT is a multimeric protein comprising a B oligomer (subunits $2-$5) that binds to receptors on target ceils, and a catalytic A subunit (S1), an ADP-ribosyltransferase that inactivates host regulatory Gproteins [5].…”

Thiol reagents are substrates for the ADP‐ribosyltransferase activity of pertussis toxin

“…Once within the cell, S-1 catalyses the transfer of ADP-ribose from NAD + to Gi, a GTP-binding regulatory protein of the adenylate cyclase complex. The toxin gene has been sequenced [2,3]. A Michaelis constant (Km) of 25/zM has been measured for NAD + in the NAD glycohydrolase reaction catalysed by the toxin, and generally .…”

“…Once within the cell, S-1 catalyses the transfer of ADP-ribose from NAD + to Gi, a GTP-binding regulatory protein of the adenylate cyclase complex. The toxin gene has been sequenced [2,3].…”