Identification and Characterization of an Azotobacter vinelandil Type I Secretion System Responsible for Export of the AIgE-Type Mannuronan C-5-Epimerases

• Published in: Journal of bacteriology Vol. 188; no. 15; p. 5551
• Main Authors: Gimmestad, Martin, Steigedal, Magnus, Ertesvåg, Helga, Moreno, Soledad
• Format: Journal Article
• Language: English
• Published: Washington American Society for Microbiology 01.08.2006
• Subjects: Bacteria; Bacteriology; Binding sites; Genes; Mutation; Proteins
• ISSN: 0021-9193; 1098-5530

Alginate is a linear copolymer of ß-D-mannuronic acid and its C-5-epimer, α-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 (A1gE1 to A1gE7) 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 (≤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. [PUBLICATION ABSTRACT]