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J. Bacteriol., May 1995, 2497-2504, Vol 177, No. 9
Copyright © 1995, American Society for Microbiology

Interaction of the Neisseria gonorrhoeae PilA protein with the pilE promoter involves multiple sites on the DNA

CG Arvidson and M So
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201-3098, USA.

PilA is the putative DNA-binding component of a two-component system that regulates transcription of the pilin expression locus (pilE) of Neisseria gonorrhoeae. Here we report the purification of the PilA protein and characterization of its DNA-binding activity. PilA was overproduced in Escherichia coli with an isopropyl-beta-D- thiogalactopyranoside (IPTG)-inducible expression vector. Cell extracts were prepared by sonication and fractionated by anion-exchange chromotography, followed by dye affinity chromatography with Cibacron Blue. Proteins were eluted by using a gradient of KCl, and PilA- containing fractions were identified by immunoblot analysis with a polyclonal anti-PilA antiserum. Purified PilA was judged to be > 90% pure, as determined by Coomassie blue staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PilA purified in this manner was used to develop a gel retardation assay with a 301-bp fragment containing the pilE promoter (PpilE) and upstream sequences as a probe. A fragment of similar size containing the E. coli aroH promoter was used as a negative control. Competition experiments using a 100- to 1,000-fold excess of unlabelled DNA fragments confirmed the specificity of PilA binding to the pilE promoter. To localize the PilA binding site within the 301-bp PpilE fragment, stepwise deletions were generated by PCR and the fragments were examined in the gel shift assay. The results of these experiments show that there are two regions upstream of PpilE that are required for binding by PilA.(ABSTRACT TRUNCATED AT 250 WORDS)

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