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Journal of Bacteriology, January 1999, p. 68-77, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cloning and Expression of Three New Azotobacter vinelandii Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases

Britt Iren Glærum Svanem,1 Gudmund Skjåk-Bræk,2 Helga Ertesvåg,1 and Svein Valla1,*

UNIGEN Center for Molecular Biology1 and Department of Biotechnology,2 Norwegian University of Science and Technology, N-7005 Trondheim, Norway

Received 17 August 1998/Accepted 26 October 1998

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously been reported. The corresponding proteins catalyze the Ca2+-dependent polymer-level epimerization of beta -D-mannuronic acid to alpha -L-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6, algE7, and algY. All three genes were sequenced and expressed in Escherichia coli. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5'-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5' part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.

* Corresponding author. Mailing address: Unigen Center of Molecular Biology, Medisinsk Teknisk Senter, N-7005 Trondheim, Norway. Phone: 47 73598680. Fax: 47 3598705. E-mail: svein.valla{at}unigen.ntnu.no.

Journal of Bacteriology, January 1999, p. 68-77, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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