Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade.

AUTOR(ES)

Uhl, M A

RESUMO

Expression of adhesins, toxins, and other virulence factors of Bordetella pertussis is under control of the BvgA and BvgS proteins, members of a bacterial two-component signal transduction family. BvgA bears sequence similarity to regulator components, whereas BvgS shows similarity to both sensor and regulator components. BvgA and the cytoplasmic portion of BvgS ('BvgS) were overexpressed and purified. 'BvgS autophosphorylated with the gamma-phosphate from [gamma-32P]ATP and phosphorylated BvgA. Kinetic analysis indicated that BvgA receives its phosphate from 'BvgS. Mutations in the transmitter, receiver, and C-terminal domains of BvgS were tested for activation of a BvgAS-dependent fhaB::lacZ reporter fusion in vivo and for autophosphorylation and phosphotransfer to BvgA in vitro. All mutations abolished activation of the fhaB::lacZ fusion. A point mutation in the transmitter (H729Q) prevented autophosphorylation of 'BvgS. In contrast to other characterized sensor proteins, autophosphorylation also required sequences in the 'BvgS receiver and C-terminal domains. A 'BvgS receiver point mutation (D1023N) had the novel phenotype of being able to autophosphorylate but unable to transfer the phosphate to BvgA. Autophosphorylation activity of the D1023N mutant protein was kinetically and chemically indistinguishable from wild-type 'BvgS despite an uncoupling of phosphotransfer from autophosphorylation. 'BvgS was shown to contain primarily amidyl phosphate and BvgA an acyl phosphate linkage. We present a model for a phosphorelay controlling virulence gene expression in B. pertussis.

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