title: Identification and characterization of an Azotobacter vinelandii type I secretion system responsible for export of the AlgE-type mannuronan C-5-epimerases.
abstract: Alginate is a linear copolymer of beta-d-mannuronic acid and its C-5-epimer, alpha-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (< or =2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.
authors.affiliation: Department of Biotechnology, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, Norway.
authors.forename: Martin
authors.initials: M
authors.lastname: Gimmestad
authors.forename: Magnus
authors.initials: M
authors.lastname: Steigedal
authors.forename: Helga
authors.initials: H
authors.lastname: Ertesvåg
authors.forename: Soledad
authors.initials: S
authors.lastname: Moreno
authors.forename: Bjørn Erik
authors.initials: BE
authors.lastname: Christensen
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Espín
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
mesh_terms: D016948:Azotobacter vinelandii / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000378:metabolism; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D010802:Phylogeny
chemical_list: D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase