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Proc Natl Acad Sci U S A 2015 Jan 26 [Epub ahead of print]

Bordetella pertussis fim3 gene regulation by BvgA: Phosphorylation controls the formation of inactive vs. active transcription complexes.

Boulanger A, Moon K, Decker KB, Chen Q, Knipling L, Stibitz S, Hinton DM


Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA approximately P) state at Pfim3, the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA approximately P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3 activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3 activation once the signal for BvgA phosphorylation is removed.

Category: Journal Article
PubMed ID: #25624471 DOI: 10.1073/pnas.1421045112
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2015-01-28 Entry Last Modified: 2015-02-02