Membrane Topology of Outer Membrane Protein AlgE, Which Is Required for Alginate Production in Pseudomonas aeruginosa

Abstract

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa secretes a viscous extracellular polysaccharide, called alginate, as a virulence factor during chronic infection of patients with cystic fibrosis. In the present study, it was demonstrated that the outer membrane protein AlgE is required for the production of alginate in P. aeruginosa. An isogenic marker-free algE deletion mutant was constructed. This strain was incapable of producing alginate but did secrete alginate degradation products, indicating that polymerization occurs but that the alginate chain is subsequently degraded during transit through the periplasm. Alginate production was restored by introducing the algE gene. The membrane topology of the outer membrane protein AlgE was assessed by site-specific insertions of FLAG epitopes into predicted extracellular loop regions.

Structural basis for alginate secretion across the bacterial outer membrane

“…AlgE forms a monomeric 18-stranded antiparallel β-barrel with the register of the β-strands being in reasonable agreement with previously reported topology models (20,23) (Fig. S1).…”

“…As reported previously, the algE mutant was nonmucoid and produced no detectable levels of alginate (Fig. 4C) (23). When the algE gene was introduced on a plasmid, wild-type levels of alginate production were restored.…”

Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

“…1). Hay et al have previously demonstrated that insertion of this epitope in loop 6 does not affect alginate production in P. aeruginosa (19). Using this approach, we investigated the correlation between the number of factories and alginate production level, their dependence on the production of alginate precursor, and the spatial distribution pattern of alginate factories on the cell surface.…”

“…According to these findings, AlgX plays a crucial role in the localization of the porin AlgE (21). The absence of any of the scaffold proteins AlgG, AlgX, AlgK, or AlgE in a complex leads to release of alginate in the periplasm, where it is degraded by AlgL, resulting in a lower polymer yield (1,(16)(17)(18)(19)(20)24).…”

“…Several studies have indicated that the newly synthesized polymannuronate chain needs to be translocated across the periplasm with the aid of a protein scaffold composed of Alg8, Alg44, AlgG, AlgX, AlgK, and AlgE (17)(18)(19)(20)(21)(22)(23). AlgK is a periplasmic lipoprotein bound to the outer membrane and is important, but not essential, for the localization of the porin AlgE.…”