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.