Abstract
Pseudomonas aeruginosa strains causing chronic pulmonary infections
in cystic fibrosis patients produce high levels of alginate, an exopolysaccharide
that confers a mucoid phenotype. Alginate is a linear polymer of
d-mannuronate (M) and variable amounts of its C-5-epimer, l-guluronate
(G). AlgG is a periplasmic C-5-epimerase that converts poly d-mannuronate
to the mixed M+G sequence of alginate. To understand better the role
and mechanism of AlgG activity, a mutant was constructed in the mucoid
strain FRD1 with a defined non-polar deletion of algG. Instead of
producing poly mannuronate, the algG deletion mutant secreted dialysable
uronic acids, as does a mutant lacking the periplasmic protein AlgK.
High levels of unsaturated ends and the nuclear magnetic resonance
spectroscopy pattern revealed that the small, secreted uronic acids
were the products of extensive polymer digestion by AlgL, a periplasmic
alginate lyase co-expressed with AlgG and AlgK. Thus, AlgG is bifunctional
with (i) epimerase activity and (ii) a role in protecting alginate
from degradation by AlgL during transport through the periplasm.
AlgK appears to share the second role. AlgG and AlgK may be part
of a periplasmic protein complex, or scaffold, that guides alginate
polymers to the outer membrane secretin (AlgE). To characterize the
epimerase activity of AlgG further, the algG4 allele of poly mannuronate-producing
FRD462 was shown to encode a protein lacking only the epimerase function.
The sequence of algG4 has a Ser-272 to Asn substitution in a serine-threonine-rich
and conserved region of AlgG, which revealed a critical residue for
C-5-epimerase activity.
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