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Journal of Bacteriology, August 2009, p. 4845-4853, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00455-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of Three New Azotobacter vinelandii Alginate Lyases, One of Which Is Involved in Cyst Germination{triangledown}

Martin Gimmestad, Helga Ertesvåg,* Tonje Marita Bjerkan Heggeset,§ Olav Aarstad, Britt Iren Glærum Svanem, and Svein Valla

Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway

Received 3 April 2009/ Accepted 26 May 2009

Alginates are polysaccharides composed of 1-4-linked β-D-mannuronic acid and {alpha}-L-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-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 Ca2+. 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.

* Corresponding author. Mailing address: Department of Biotechnology, Sem Sælandsvg 6-8, NTNU, N-7491 Trondheim, Norway. Phone: (47)73598678 Fax: (47)73591283. E-mail: helga.ertesvag{at}biotech.ntnu.no

{triangledown} Published ahead of print on 26 May 2009.

§ Present address: SINTEF Materials and Chemistry, Department of Biotechnology, N-7465 Trondheim, Norway.

Journal of Bacteriology, August 2009, p. 4845-4853, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00455-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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