Molecular Microbiology (1996) zyxwvutsrq 21 zyxwvutsrqp (3), 557-565 Differential binding of BvgA to two classes of virulence genes of zyxwv Bordetella pertussis directs promoter selectivity by RNA polymerase Tao Zu,+ Riccardo Manetti, Rino Rappuoli and Vincenzo Scarlato* zyxwvutsrq Department of Molecular Biology, IRIS, Chiron-Biocine lmmunobiological Research Institute in Siena, Via Fiorentina 1, 53 100 Siena, Italy. Summary Transcription of virulence genes of Bordetella pertus- sis is co-ordinately regulated by the BvgA and BvgS proteins, which are members of the two-component family of bacterial signal-transduction proteins. BvgS is the transmembrane sensor and BvgA the transcrip- tional regulator. By gel mobility shift assays we demonstrate that phosphorylated BvgA (BvgA E P) forms distinct complexes with the filamentous haemag- glutinin (PFHA) promoter DNA at different BvgAZ P: DNA ratios. DNase I protection analyses show that phosphorylation of BvgA not only enhances affinity of the protein for the binding sites of the PFHn and bvgPI promoters, but it extends significantly the bound region towards position -35 of these promoters. Conversely, a 10-fold higher amount of BvgAEP is required for binding to a large DNA region, from -168 to -60, of the pertussis toxin (PtOx) promoter sequence. These findings suggest that the molecular interaction of BvgA % zyxwvutsrqp P with the P , , , promoter is different from its interaction with the PFHn and bvgP, promoters. The 0' Escherichia coli RNA polymerase (RNP) does not bind to the bvgregulated promoters. However, follow- ing the formation of a BvgA % P-promoter complex, the zyxwvutsrqpo E. coli RNP specifically recognizes and binds to the bvgregulated promoters. Thus, BvgA RZ P exerts its action at the level of promoter recognition by direct- ing promoter selectivity by RNP. Introduction Expression of virulence genes of Bordetella pertussis, the human pathogen which causes whooping cough, is Received 25 March, 1996; revised 17 May, 1996; accepted 18 May, 1996. ?Present address: Department of Biological Sciences, Univer- sity of California at Santa Barbara, Santa Barbara, California 931 06, USA. *For correspondence. E-mail Scarlato@ iris02.biocine.it; Tel. (577) 243480; Fax (577) 243564. zyxwvutsrq 0 1996 Blackwell Science Ltd co-ordinately regulated by a sensory transduction system encoded by the bvgAS locus (Arico et a/., 1989; Stibitz and Yang, 1991 ; Weiss et a/., 1983). The transcriptional activator BvgA and the sensor protein BvgS are members of the two-component family of bacterial signal-transduction proteins (Coote, 1991; Miller et a/., 1989a; Stock et a/., 1990). As suggested by phosphorelay studies of the bvg system (Uhl and Miller, 1994), communication between these two molecules is achieved by protein phosphoryla- tion. BvgS phosphorylates the transcriptional activator BvgA, which in turn activates transcription of the virulence genes. Transcriptional analysis has shown that according to environmental signals such as temperature switch from 25% to 37"C, the bvgregulated promoters can be divided into two groups: early and late (Scarlato etal., 1991 a). The early promoters are activated within a few minutes of induction (at 37'C) and include those controlling transcrip- tion of filamentous haemagglutinin (fhaB) and pili genes. The late promoters are activated hours after induction, and include those controlling the expression of pertussis toxin (pfx) and adenylate cyclase (cyaA) operons. Tran- scription at the bvg locus is controlled by four autoregu- lated promoters that allow a tight control of virulence under any growth condition (Scarlato et a/., 1990). One of these bvg promoters, PI, is a typical early promoter that, following a positive signal (37C), starts to synthesize a bvg mRNA responsible for an overproduction and intra- cellular accumulation of the transcriptional activator BvgA (Scarlato et a/., 1990; 1991a). After 4 h of temperature induction the concentration of BvgA increases 60-fold and starts transcription from the late promoters. In addition to the temporal activation of virulence gene promoters in 6. pertussis, early and late promoters can be distinguished by a number of other features. In Escherichia coli, the PI and PFHn early promoters are regulated similarly to those in zyxwvut B. pertussis, under the control of the bvg locus, while the Ptox and cyaA late promoters were found to be non- functional (Goyard and Ullmann, 1991; Miller et a/., 1989b; Roy etal., 1989). Gel mobility shift and footprinting experiments, using crude protein extracts from E, coliover- expressing BvgA, showed that this protein binds to the early promoter sequences, while binding to the late pro- moters was not demonstrated (Boucher et a/., 1994; Roy and Falkow, 1991).These observationsled to the hypothesis