Role of the Pseudomonas fluorescens Alginate Lyase (AlgL) in Clearing the Periplasm of Alginates Not Exported to the Extracellular Environment

Abstract

Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

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.…”

A Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique, pH-regulated Substrate Specificity

“…In Mycobacterium tuberculosis, secreted HA lyases generate low molecular weight sugars from HA that serve as carbon source for bacteria to replicate in deep tissue infections (14). In the case of P. aeruginosa biofilm formation, the periplasmic alginate lyase AlgL regulates the chain length and concentration of alginate in the periplasm during secretion (15). Loss of AlgL results in cell lysis for mucoid strains of P. aeruginosa due to alginate accumulation in the periplasm (16), and AlgL overexpression leads to truncation of alginate to short-chain polysaccharides unable to form extended extracellular aggregates.…”

Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation

“…Deletion of algL presumably prevents complex assembly, resulting in polymer accumulation in the periplasm and cell rupture. To our knowledge, this is the only example where the absence of a polysaccharide-cleaving enzyme results in a lethal phenotype because deletion or mutation of catalytic residues of many GHs either impairs polysaccharide export or does not affect biosynthesis (20,(22)(23)(24)(25). This difference may be attributed to the fact that each mannuronate residue in alginate carries a Ϫ1 charge, and retention of this anionic polymer in the periplasm would be detrimental to the proton motive force by reducing the membrane potential.…”

“…PslG Is Not Necessary for Psl Biosynthesis but Overexpression Leads to Defects in Biofilm Formation-Previous genetic deletions of pslG and genes encoding other hydrolytic enzymes have demonstrated that they are required for exopolysaccharide biosynthesis (8,20,(22)(23)(24)(25). To determine whether the glycoside hydrolase activity of PslG is required for Psl biosynthesis, chromosomal point mutations, pslG E165Q , pslG E276Q , and pslG…”

“…Examples include Escherichia coli BcsZ, Pseudomonas fluorescens WssD, P. aeruginosa AlgL, Listeria monocytogenes PssZ, the N-terminal domain of P. aeruginosa PelA, and the C-terminal domain of E. coli PgaB involved in cellulose, acetylated cellulose, alginate, the L. monocytogenes exopolysaccharide, Pel, and PNAG biosynthesis, respectively (16 -21). The function of putative polysaccharide-degrading enzymes in exopolysaccharide biosynthetic operons is not intuitive or well defined; however, genetic deletions indicate that many are obligatory for polysaccharide biosynthesis (20,(22)(23)(24)(25).…”