title: Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination.
abstract: Alginates are polysaccharides composed of 1-4-linked beta-D-mannuronic acid and alpha-L-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a beta-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca(2+). All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Martin
authors.initials: M
authors.lastname: Gimmestad
authors.forename: Helga
authors.initials: H
authors.lastname: Ertesvåg
authors.forename: Tonje Marita Bjerkan
authors.initials: TM
authors.lastname: Heggeset
authors.forename: Olav
authors.initials: O
authors.lastname: Aarstad
authors.forename: Britt Iren Glaerum
authors.initials: BI
authors.lastname: Svanem
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
mesh_terms: D000464:Alginates / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; C007896:guluronic acid; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase