The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis

Francis Wolfram,Alison M Berezuk,Holly M Jacobs,Roland Pfoh,Gregory B Whitfield,Mara C Goodyear,P Lynne Howell,Cezar M Khursigara,Matthew R Parsek,Steven S.Y Wong,Allison K Guitor,Andreea A Gheorghita

doi:10.1016/j.jbc.2021.101560

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein’s role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.

Highlights

Aureus, influencing cystic fibrosis (CF) patient outcomes as co-infection is associated with decreased lung function (27–29)
Our disordered in the structure (Fig. 1). While this structure of wild-type (WT) P. aeruginosa AlgL in mannuronic acid (ManA) identifies the location of the active site, we Journal Pre-proof complex with ManA, its comparison with other bacterial alginate lyases, and in vitro enzyme kinetic analyses have enabled the identification of active site residues important for alginate binding and catalysis
In a genetically engineered strain where alginate biosynthesis can be controlled using arabinose, we demonstrate that absence of algL or mutation of key catalytic residues is detrimental for P. aeruginosa growth during alginate biosynthesis and results in abnormal cellular morphology

Summary

INTRODUCTION

In alginate biosynthesis has been observed in a Biofilms are highly structured communities of P. aeruginosa murine burn wound model bacterial cells embedded in a self-produced matrix demonstrating the polymer’s importance in biofilm (1, 2). The alg operon encodes a periplasmic producing three distinct exopolysaccharides as part lyase, AlgL Characterization of this enzyme in of its biofilm matrix: Pel, Psl, and alginate. Alginate is part of a multiprotein complex with AlgG, AlgX, produced when P. aeruginosa converts to a mucoid and AlgK and that it assists in transporting the polymer across the outer membrane (47) This reveals that this (α/α)n spatial arrangement has been contrasts with more recent studies that suggest reported previously in alginate lyases from the. Our disordered in the structure (Fig. 1) While this structure of wild-type (WT) P. aeruginosa AlgL in ManA identifies the location of the active site, we Journal Pre-proof complex with ManA, its comparison with other bacterial alginate lyases, and in vitro enzyme kinetic analyses have enabled the identification of active site residues important for alginate binding and catalysis.

RESULTS

Findings

D Secreted