A phage promoter can substitute for the <i>pilE</i> G4-associated sRNA promoter.
2013-01-17T01:51:30Z (GMT) by
<p><b>A. </b><i>In vitro</i> transcription. Shown is a 14% denaturing polyacrylamide gel containing T7 RNA polymerase-transcribed RNA products from a template containing the minimal T7 promoter element. The RNA ladder is labeled in bases. The higher molecular weight band is most likely an alternative start site directed by the T7 polymerase. <b>B.</b> The T7 promoter/T7 RNA polymerase strain. T7 RNA polymerase was cloned downstream of a dual <i>taclac</i> promoter which allows induction with IPTG <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003074#ppat.1003074-Mehr1" target="_blank"></a>. The construct was transformed into a strain where the <i>pilE</i> G4 sRNA −10 promoter element was replaced with a minimal T7 promoter and inserted into the neisserial intergenic complementation site (NICS). [A triangle indicates a transposon (TN) encoding kanamycin resistance and was used as a marker for transformation to create the sRNA T7 promoter mutant]. <b>C.</b> Colony morphology of the T7 promoter and/or T7 polymerase strains. Colonies were grown for 46 hours with IPTG. The parental strain shows an Av phenotype, a strain expressing only T7 at the NICS (NICS T7 Pol.) has an Av phenotype, a strain where the G4 sRNA −10 element is replaced by the minimal T7 promoter (T7 Prom.) shows an Av-deficient (Avd) phenotype that is rescued with the expression of T7 polymerase (T7 Prom./NICS T7 Pol.). <b>D.</b> Kinetic pilus-dependent colony phase variation assay. Expression of T7 polymerase at the NICS (NICS T7 Pol.) does not affect phase variation whereas replacement of the G4 sRNA promoter with a minimal T7 promoter (T7 Prom.) causes a decrease in phase variation which is rescued by expression of T7 polymerase at the NICS (T7 Prom./NICS T7 Pol.). <i>P</i><0.05 as determined by two-tailed Student's T-test (asterisks). Error bars represent the standard error of the mean for 10 colonies. A representative assay of <i>n</i> = 4 is shown.</p>