The dual roles of AlgG in C‐5‐epimerization and secretion of alginate polymers in Pseudomonas aeruginosa

Abstract

SummaryPseudomonas 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 mannuronateproducing 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 serinethreonine-rich and conserved region of AlgG, which revealed a critical residue for C-5-epimerase activity.

The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation

“…indicating that the degradation product might be primarily a dimer. Similar results were found in a parallel study on P. aeruginosa (22). This was surprising since it has been shown previously that when AlgL from A. vinelandii was used to completely degrade mannuronan in vitro (9), the average degree of polymerization of the endproducts was about three.…”

“…The alginates used were from Pf201 (33% G) (؋), Pf20118 (100% M) (}), Pf20118::TnKB10 (18% G) (OE), and Pf201 and Pf20118 (17% G total) (F). (1), and deletion mutants of algK have been shown to make small oligouronides with unsaturated nonreducing ends similar to those found for algG deletion reported here (22,23). Finally, AlgE has been reported to be an outer membrane pore necessary for alginate export (36).…”

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

“…earlier stage (in the periplasm) may disturb the delicate control of alginate structure needed for this species. An AlgG protein structure may still be needed since this protein has been found to display an additional role (probably structural) in protecting the newly synthesized polymer from AlgL-mediated degradation (1,21,29,38). The eex mutants do not form stable cell coats in RA1 medium.…”

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

“…When either AlgK, G or X is absent from the cell, new polymers are subjected to degradation by AlgL, an alginate lyase encoded by the algD operon (Schiller et al, 1993). Such degraded polymers are readily found in the culture supernatant as dialysable uronic acids (Jain & Ohman, 1998Jain et al, 2003;Robles-Price et al, 2004). When AlgL is absent, polymer cannot escape the periplasm, suggesting a role for AlgL in polymer transport as well as its alginate-lyase activity (Jain & Ohman, 2005).…”

“…However, they produce small fragments of alginate due to the action of AlgL, an alginate lyase co-expressed in the algD operon, which degrades newly formed polymers (Jain & Ohman, 1998Jain et al, 2003;Robles-Price et al, 2004). AlgK, AlgG, AlgX and AlgL may form a periplasmic scaffold to bring newly synthesized polymers to the outer-membrane porin, AlgE, and protect the polymer from degradation by AlgL (Jain & Ohman, 2005).…”