AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa.
Alginate, an exopolysaccharide produced by Pseudomonas aeruginosa, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene, algX, within the alginate biosynthetic gene cluster on the P. aeruginosa chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of algX in P. aeruginosa strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting algXDelta::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The algXDelta::Gm mutant was restored to the mucoid phenotype with wild-type P. aeruginosa algX provided on a plasmid. The algXDelta::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.
Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, USA.
Antonette
A
Robles-Price
Thiang Yian
TY
Wong
Håvard
H
Sletta
Svein
S
Valla
Neal L
NL
Schiller
D000464:Alginates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D020723:Glucuronic Acid / Q000378:metabolism*; D006603:Hexuronic Acids / Q000378:metabolism*; D006801:Humans; D033903:Periplasmic Proteins / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014574:Uronic Acids / Q000737:chemistry / Q000378:metabolism
D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D033903:Periplasmic Proteins; D011108:Polymers; D014574:Uronic Acids; D020723:Glucuronic Acid