title: Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation.
abstract: The Pseudomonas aeruginosa algD gene is the first gene of an operon encoding most of the enzymes necessary for biosynthesis of the exopolysaccharide alginate. Transcriptional activation of algD results in the high-level synthesis of alginate, an important P. aeruginosa virulence factor with antiphagocytic and adherence properties. Previously, we have identified a protein(s), AlgZ, expressed in mucoid P. aeruginosa CF isolates that specifically bound to sequences located 280 bp upstream of the algD promoter. Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show that AlgZ was an activator of algD transcription. In the current study, the monomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electroelution 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 AlgZ, was devised, the protein identified and a partial amino-terminal sequence obtained. Using the P. aeruginosa Genome Project database, a complete sequence was obtained, and algZ was cloned and expressed in Escherichia coli. Expression of algZ was sufficient for the observed AlgZ DNA binding previously observed from extracts of P. aeruginosa. A protein database search revealed that AlgZ is homologous to the Mnt and Arc repressors of the ribbon-helix-helix family of DNA-binding proteins. An algZ deletion mutant was constructed in the mucoid CF isolate FRD1. The resulting strain was non-mucoid and exhibited no detectable algD transcription. As an indirect role in transcription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hypothesis that both AlgZ and the response regulator AlgR are involved in direct contact with RNA polymerase containing the alternative sigma factor, AlgT. The cloning of algZ is a crucial step in determining the mechanism of algD activation.
authors.affiliation: Department of Microbiology and Immunology, Medical Center Boulevard, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Baynham
authors.forename: A L
authors.initials: AL
authors.lastname: Brown
authors.forename: L L
authors.initials: LL
authors.lastname: Hall
authors.forename: D J
authors.initials: DJ
authors.lastname: Wozniak
mesh_terms: D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008969:Molecular Sequence Data; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006603:Hexuronic Acids; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D020723:Glucuronic Acid; D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase