title: Insights into the assembly of the alginate biosynthesis machinery in Pseudomonas aeruginosa.
abstract: Pseudomonas aeruginosa is an opportunistic pathogen of particular significance to cystic fibrosis patients. This bacterium produces the exopolysaccharide alginate, which is an indicator of poor prognosis for these patients. The proteins required for alginate polymerization and secretion are encoded by genes organized in a single operon; however, the existence of internal promoters has been reported. It has been proposed that these proteins form a multiprotein complex which extends from the inner to outer membrane. Here, experimental evidence supporting such a multiprotein complex was obtained via mutual stability analysis, pulldown assays, and coimmunoprecipitation. The impact of the absence of single proteins or subunits on this multiprotein complex, i.e., on the stability of potentially interacting proteins, as well as on alginate production was investigated. Deletion of algK in an alginate-overproducing strain, PDO300, interfered with the polymerization of alginate, suggesting that in the absence of AlgK, the polymerase and copolymerase subunits, Alg8 and Alg44, are destabilized. Based on mutual stability analysis, interactions between AlgE (outer membrane), AlgK (periplasm), AlgX (periplasm), Alg44 (inner membrane), Alg8 (inner membrane), and AlgG (periplasm) were proposed. Coimmunoprecipitation using a FLAG-tagged variant of AlgE further demonstrated its interaction with AlgK. Pulldown assays using histidine-tagged AlgK showed that AlgK interacts with AlgX, which in turn was also copurified with histidine-tagged Alg44. Detection of AlgG and AlgE in PAO1 supported the existence of internal promoters controlling expression of the respective genes. Overall experimental evidence was provided for the existence of a multiprotein complex required for alginate polymerization and secretion.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Zahid U
authors.initials: ZU
authors.lastname: Rehman
authors.forename: Yajie
authors.initials: Y
authors.lastname: Wang
authors.forename: M Fata
authors.initials: MF
authors.lastname: Moradali
authors.forename: Iain D
authors.initials: ID
authors.lastname: Hay
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D000464:Alginates; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D020723:Glucuronic Acid / Q000096:biosynthesis / Q000235:genetics; D006603:Hexuronic Acids; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D058105:Polymerization; D011401:Promoter Regions, Genetic; D025941:Protein Interaction Mapping; D055550:Protein Stability; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
chemical_list: C085500:Alg44 protein, Pseudomonas aeruginosa; C085499:Alg8 protein, Pseudomonas aeruginosa; C104798:AlgK protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D020723:Glucuronic Acid