Abstract
Carbohydrate polymers are industrially and medically important. For instance, a polysaccharide, alginate (from seaweed), is widely used in food, textile and pharmaceutical industries. Certain bacteria also produce alginate through membrane spanning multi-protein complexes. Using Pseudomonas aeruginosa as a model organism, we investigated the biological function of an alginate degrading enzyme, AlgL, in alginate production and biofilm formation. We showed that AlgL negatively impacts alginate production through its enzymatic activity. We also demonstrated that deletion of AlgL does not interfere with polymer length control, epimerization degree or stability of the biosynthesis complex, arguing that AlgL is a free periplasmic protein dispensable for alginate production. This was further supported by our protein-stability and interaction experiments. Interestingly, over-production of AlgL interfered with polymer length control, suggesting that AlgL could be loosely associated with the biosynthesis complex. In addition, chromosomal expression of algL enhanced alginate O-acetylation; both attachment and dispersal stages of the bacterial biofilm lifecycle were sensitive to the level of O-acetylation. Since this modification also protects the pathogen against host defences and enhances other virulence factors, chromosomal expression of algL could be important for the pathogenicity of this organism. Overall, this work improves our understanding of bacterial alginate production and provides new knowledge for alginate production and disease control.
Highlights
An alginate degrading enzyme, AlgL, is encoded within the alginate biosynthesis gene cluster
Moradali et al.[14] showed that the molecular mass of alginate was reduced by epimerization, while it was increased by acetylation, the role of AlgL and its lyase activity in controlling polymer length, composition and alginate yield is still unknown
When a catalytically inactive variant of algL was overexpressed in the algL mutant, PDO300ΔalgL(pHERD20T:algLH202A), alginate yield was increased by three-fold compared to wild type (WT) strain harboring an empty vector, PDO300(pHERD20T)
Summary
An alginate degrading enzyme, AlgL, is encoded within the alginate biosynthesis gene cluster. Even though the exact biological function of AlgL remains undetermined, two preliminary models for its role in alginate biosynthesis have been proposed[33,36] These models suggest that AlgL undertakes a maintenance role in the periplasm by degrading alginate that has been misguided due to inefficient translocation/secretion, deletion of a subunit or destabilization of the complex. They disagree on whether AlgL is part of the biosynthesis apparatus. A better understanding of biological function of alginate and AlgL in biofilm attachment and dispersal would help inform future strategies utilizing alginate degrading enzymes for combating P. aeruginosa infections. The knowledge generated in this study is of importance for development of approaches for homogenous alginate production and disease treatment
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