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

Abstract

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-␤-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 (P pilE ) 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 P pilE 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 P pilE that are required for binding by PilA. Taken together, these data indicate that while PilA binds specifically to the upstream region of the pilE gene, this interaction is complex and likely involves multiple regions of this DNA sequence.Neisseria gonorrhoeae is a major sexually transmitted pathogen that infects only humans. The gram-negative diplococcus normally infects cells of the urogenital tract, although it can also infect other human tissues such as the conjunctiva and pharynx. Experimental infection of Fallopian tube organ cultures shows that the bacteria attach to nonciliated epithelial cells of the mucosa, invade these cells, and transcytose to the subepithelial matrix (21). Attachment of the bacteria to epithelial cells occurs via adhesins including pili and the opacity protein (Opa or PII;17,44). Pili are considered a major virulence factor, because only piliated variants produce infection in healthy volunteers (14,45).The gonococcal chromosome harbors numerous variant pil genes that participate in phase and antigenic variation of these proteins. In vitro, pilin antigenic variation can occur via DNA transformation and recombination or gene conversion involving silent pil loci (pilS) and the pilin expression site (pilE) (7, 36). Phase variation can also occur by this mechanism, i.e., if a recombination event results in an internal frameshift, P ϩ (piliated)-to-P Ϫ (nonpiliated) and P Ϫ -to-P ϩ transitions can occur (1a). Recombination can also result in deletion of all or part of the transcriptional or translational signals, ...

Role for both DNA and RNA in GTP Hydrolysis by the Neisseria gonorrhoeae Signal Recognition Particle Receptor

“…Whether or not this is truly unique to neisseriae remains to be seen. Our results also provide some insight into the previously puzzling observation that PilA binds to pilE promoter DNA in a sequence-specific manner (2). We now know that PilA, rather than functioning as a transcriptional regulator of gonococcal pilin biosynthesis, is part of the SRP protein targeting pathway (1).…”

“…PilA exhibits anomalous sequence-specific DNA-binding activity (2). At the time the DNA-binding activity was identified, PilA was thought to be a transcriptional regulator that bound to promoter DNA to activate and/or repress transcription of the associated gene (54).…”

“…We have also shown that PilA has sequence-specific DNA-binding activity in vitro (2). Up to this point, we have not been able to reconcile the DNAbinding capability of PilA with its role in protein targeting.…”

The Neisseria Transcriptional Regulator PilA Has a GTPase Activity

“…We have previously demonstrated that PilA is a DNA-binding protein that binds specifically to a region 5Ј to the pilE1 promoter in a complex manner that may involve looping of the DNA (12). The PilA in these experiments was purified from a strain lacking pilB and was presumed to be unphosphorylated.…”

“…1). The SRP54 proteins have two domains: an "M" domain, which interacts with the signal peptide of nascent proteins as well as to the 7 S (4.5 S in E. coli) RNA, and a "G" domain, which has the GTPase activity required for the interaction of the complex with the docking protein (19 Protein Purification-PilA was purified as described (12) with the following modification: Cibacron Blue 3GA-Agarose (Sigma) was used as the dye affinity resin, and PilA was batch-eluted from this resin at 0.3 M KCl. Protein concentration was determined using the Bradford method (Bio-Rad).…”