title: 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-terminal amino 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 algE deletion mutant PDO300ΔalgE(miniCTX) showed the periplasmic alginate biosynthesis proteins AlgK and AlgX were completely destabilized, while the copy number of the inner membrane c-di-GMP receptor Alg44 was reduced. Chromosomal integration of algE 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.
authors: Institute of Fundamental Sciences and MacDiarmid Institute for Advanced Materials and Nanotechnology, Massey University, Palmerston North, New Zealand.
authors: Zahid U
authors: ZU
authors: Rehman
authors: Bernd H A
authors: BH
authors: Rehm
mesh_terms: D000464:Alginates; D001692:Biological Transport; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D005816:Genetic Complementation Test; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D037521:Virulence Factors; D020723:Glucuronic Acid