Dual roles of Pseudomonas aeruginosa AlgE in secretion of the virulence factor alginate and formation of the secretion complex

Abstract

AlgE is a monomeric 18-stranded β-barrel protein required for secretion of the extracellular polysaccharide alginate in Pseudomonas aeruginosa. To assess the molecular mechanism of alginate secretion, AlgE was subjected to site-specific and FLAG epitope insertion mutagenesis. Except for β-strands 6 and 10, epitope insertions into the transmembrane β-strands abolished localization of AlgE to the outer membrane. Interestingly, an epitope insertion into β-strand 10 produced alginate and was only detectable in outer membranes isolated from cells grown on solid media. The deletion of nine C-terminalamino acid residues destabilized AlgE. Replacement of amino acids that constitute the highly electropositive pore constriction showed that individual amino acid residues have a specific function in alginate secretion. Two of the triple mutants(K47E-R353A-R459E and R74E-R362A-R459E) severely reduced alginate production. Mutual stability analysis using the algEdeletion mutant PDO300-algE(miniCTX) showed the periplasmic alginate biosynthesis proteins AlgK and AlgX were completelydestabilized, while the copy number of the inner membrane c-di-GMP receptor Alg44 was reduced. Chromosomal integration ofalgE restored AlgK, AlgX, and Alg44, providing evidence for a multiprotein complex that spans the cell envelope. Periplasmic turn 4 of AlgE was identified as an important region for maintaining the stability of the putative multiprotein complex. © 2013, American Society for Microbiology.