Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ (alginate and motility regulator Z).

Transcriptional activation of the Pseudomonas aeruginosa algD gene results in high-level synthesis of the capsular polysaccharide alginate, an important P. aeruginosa virulence factor expressed in cystic fibrosis (CF) patients with chronic pulmonary disease. In this study, electrophoretic mobility-shift assays were used to identify a novel protein (AlgZ), which binds specifically to a sequence located 280 bp upstream of the algD promoter. While AlgZ-binding activity did not require the response regulators AlgB or AlgR, expression of AlgZ was found to be absolutely dependent on the alternative sigma factor AlgT. Electrophoretic mobility-shift assays and copper-phenanthroline footprinting localized AlgZ binding to a 36 bp algD region, which includes several helical repeats. A collection of alginate-producing (mucoid) and non-mucoid P. aeruginosa strains, derived from CF patients, was characterized for AlgZ-binding activity. In all cases, AlgZ binding to algD sequences was observed when extracts derived from mucoid P. aeruginosa CF isolates were examined. However, this binding activity was not present when extracts from non-mucoid P. aeruginosa CF isolates were tested. Oligonucleotide mutagenesis was employed to create an algD allele with a 4 bp mutation in the predicted AlgZ-binding site (algD38) and a heterologous substitution allele (algD40), in which the entire AlgZ-binding site was replaced with a non-specific DNA sequence of identical size. When the algD38 mutation was cloned into an algD-cat transcriptional fusion, this resulted in a 28-fold reduction in algD expression, whereas the algD40 mutation abolished algD transcription, indicating that AlgZ acts as an activator of algD transcription. These results support the hypothesis that activation of algD involves the formation of a high-order looped structure allowing for multivalent contacts between AlgZ, AlgR and RNA polymerase containing the alternative sigma factor AlgT. Characterization of the molecular details of algD activation will provide insights into the control of other prokaryotic and eukaryotic promoters that utilize multiple activators.