The Azotobacter vinelandii AlgE mannuronan C-5-epimerase family is essential for the in vivo control of alginate monomer composition and for functional cyst formation.
The industrially widely used polysaccharide alginate is a co-polymer of beta-D-mannuronic acid and alpha-L-guluronic acid (G), and the G residues originate from a polymer-level epimerization process catalysed by mannuronan C-5-epimerases. In the genome of the alginate-producing bacterium Azotobacter vinelandii genes encoding one periplasmic (AlgG) and seven secreted such epimerases (AlgE1-7) have been identified. Here we report the generation of a strain (MS163171) in which all the algE genes were inactivated by deletion (algE1-4 and algE6-7) or interruption (algE5). Shake flask-grown MS163171 produced a polymer containing less than 2% G (algG still active), while wild-type alginates contained 25% G. Interestingly, addition of proteases to the MS163171 growth medium resulted in a strong increase in the chain lengths of the alginates produced. MS163171 was found to be unable to form functional cysts, which is a desiccation-resistant differentiated form developed by A. vinelandii under certain environmental conditions. We also generated mutants carrying interruptions in each separate algE gene, and a strain containing algE5 only. Studies of these mutants indicated that single algE gene inactivations, with the exception of algE3, did not affect the fractional G content much. However, for all strains tested the alginate composition varied somewhat as a response to the growth conditions.
Steigedal
Magnus
M
Department of Biotechnology, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway.
Sletta
Håvard
H
Moreno
Soledad
S
Maerk
Mali
M
Christensen
Bjørn E
BE
Bjerkan
Tonje
T
Ellingsen
Trond Erling
TE
Espìn
Guadalupe
G
Ertesvåg
Helga
H
Valla
Svein
S
D000464:Alginates / Q000737:chemistry* / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000737:chemistry / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000378:metabolism / Q000502:physiology*; D017353:Gene Deletion; D005798:Genes, Bacterial; D020723:Glucuronic Acid; D006603:Hexuronic Acids / Q000378:metabolism*
D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid; C008324:mannuronic acid; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase