INTERACTION OF THE NEISSERIA-GONORRHOEAE PILA PROTEIN WITH THE PILE PROMOTER INVOLVES MULTIPLE SITES ON THE DNA

PilA is the putative DNA-binding component of a two component system t hat regulates transcription of the pilin expression locus (pilE) of Ne isseria gonorrhoeae. Here we report the purification of the PilA prote in and characterization of its DNA-binding activity. PilA was overprod uced in Escherichia coli with an isopropyl-beta-D-thiogalactopyranosid e (IPTG)-inducible expression vector. Cell extracts were prepared by s onication and fractionated by anion-exchange chromotography, followed by dye affinity chromatography with Cibacron Blue. Proteins were elute d by using a gradient of KCl, and PiLA-containing fractions were ident ified by immunoblot analysis with a polyclonal anti-PilA antiserum. Pu rified PilA was judged to be >90% pure, as determined by Coomassie blu e staining and sodium dodecyl sulfate-polyacrylamide gel electrophores is. PilA purified in this manner was used to develop a gel retardation assay with a 301-bp fragment containing the pilE promoter (P-pilE) an d 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 fragm ents confirmed the specificity of PilA binding to the pilE promoter. T o localize the PilA binding site within the 301-bp P-pilE fragment, st epwise deletions were generated by PCR and the fragments were examined in the gel shift assay. The results of these experiments show that th ere are two regions upstream of P-pilE that are required for binding b y PilA. Taken together, these data indicate that while PilA binds spec ifically to the upstream region of the pilE gene, this interaction is complex and likely involves multiple regions of this DNA sequence.