Pseudomonas aeruginosa AlgR regulates type IV pilus biosynthesis by activating transcription of the fimU-pilVWXY1Y2E operon

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC

^* To whom correspondence should be addressed. Email: dwozniak{at}wfubmc.edu.

	Abstract

The response regulator AlgR is required for Pseudomonas aeruginosa type IV pilus-dependent twitching motility, a flagella-independent mode of solid surface translocation. Prior work showed that AlgR is phosphorylated at aspartate 54 and cells expressing an AlgR variant that cannot undergo phosphorylation (AlgRD54N) lack twitching motility. However, the mechanism by which AlgR controls twitching motility is not completely understood. We hypothesized that AlgR functioned by activating genes within the pre-pilin fimU-pilVWXY1Y2E cluster that are necessary for type IV pilin biogenesis. Reverse transcriptase PCR analysis showed that the fimU-pilVWXY1Y2E genes are co-transcribed in an operon, which is under the control of AlgR. This supports prior transcriptional profiling studies of wild type and algR mutants. Moreover, expression of the fimU-pilVWXY1Y2E operon was reduced in strains expressing AlgRD54N. DNAse footprinting and electrophoretic mobility shift assays demonstrate that AlgR but not AlgRD54N bound with high affinity to two sites upstream of the fimU-pilVWXY1Y2E operon. Altogether, our findings indicate that AlgR is essential for proper pilin localization and that phosphorylation of AlgR results in direct activation of the fimU-pilVWXY1Y2E operon, which is required for the assembly and export of a functional type IV pilus.