Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation

The Pseudomonas aeruginosa algD gene is the first gene of an operon encodin g most of the enzymes necessary for biosynthesis of the exopolysaccharide a lginate, Transcriptional activation of algD results in the high-level synth esis of alginate, an important P. aeruginosa virulence factor with antiphag ocytic and adherence properties. Previously, we have identified a protein(s ), AlgZ, expressed in mucoid P. aeruginosa CF isolates that specifically bo und to sequences located 280 bp upstream of the algD promoter. Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show tha t AlgZ was an activator of algD transcription. In the current study, the mo nomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electr oelution of a protein preparation from an SDS-PAGE gel and analysis of the fractions via protein staining and electrophoretic mobility shift assays. A biochemical enrichment procedure, resulting in a 130-fold enrichment for A lgZ, was devised, the protein identified and a partial amino-terminal seque nce obtained. Using the P. aeruginosa Genome Project database, a complete s equence was obtained, and algZ was cloned and expressed in Escherichia coli , Expression of algZ was sufficient for the observed AlgZ DNA binding previ ously observed from extracts of P. aeruginosa, A protein database search re vealed that AlgZ is homologous to the Mnt and Are repressors of the ribbon- helix-helix family of DNA-binding proteins. An algZ deletion mutant was con structed in the mucoid CF isolate FRD1, The resulting strain was non-mucoid and exhibited no detectable algD transcription. As an indirect role in tra nscription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hy pothesis that both AlgZ and the response regulator AlgR are involved in dir ect contact with RNA polymerase containing the alternative sigma factor, Al gT. The cloning of algZ is a crucial step in determining the mechanism of a lgD activation.