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Journal of Bacteriology, November 2004, p. 7369-7377, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7369-7377.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

AlgX Is a Periplasmic Protein Required for Alginate Biosynthesis in Pseudomonas aeruginosa

Antonette Robles-Price,1 Thiang Yian Wong,1,{dagger} Håvard Sletta,2 Svein Valla,3 and Neal L. Schiller1*

University of California, Riverside, Riverside, California,1 SINTEF Applied Chemistry,2 Department of Biotechnology, NTNU Norwegian University of Science and Technology, Trondheim, Norway3

Received 18 May 2004/ Accepted 5 August 2004

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 algX{Delta}::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The algX{Delta}::Gm mutant was restored to the mucoid phenotype with wild-type P. aeruginosa algX provided on a plasmid. The algX{Delta}::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.

* Corresponding author. Mailing address: Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121. Phone: (909) 787-4569. Fax: (909) 787-5504. E-mail: neal.schiller{at}ucr.edu.

{dagger} Present address: The Burnham Institute, La Jolla, CA 92037.

Journal of Bacteriology, November 2004, p. 7369-7377, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7369-7377.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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