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Journal of Bacteriology, June 2002, p. 3000-3007, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3000-3007.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutant Analysis and Cellular Localization of the AlgI, AlgJ, and AlgF Proteins Required for O Acetylation of Alginate in Pseudomonas aeruginosa

Michael J. Franklin1 and Dennis E. Ohman2,3*

Department of Microbiology and Center for Biofilm Engineering, Montana State University, Bozeman, Montana,1 Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University,2 McGuire Veterans Affairs Medical Center, Richmond, Virginia3

Received 4 May 2001/ Accepted 27 February 2002

Alginate is an extracellular polysaccharide produced by mucoid strains of Pseudomonas aeruginosa that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients. Alginate is a linear polymer of D-mannuronate and L-guluronate with O-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues. The presence of O-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the algF, algJ, and algI genes are known to be essential for the addition of O-acetyl groups to alginate. To better understand the mechanism of O acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar algI, algJ, and algF deletion mutants of P. aeruginosa strain FRD1 were constructed, and each mutant produced alginate lacking O-acetyl groups. Expression of algI, algJ, or algF in trans in the corresponding mutant complemented each O acetylation defect. Random phoA (alkaline phosphatase [AP] gene) fusions to algF, algJ, and algI were constructed. All in-frame fusions to algF and algJ had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm. Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction. An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide. AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains. These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for O acetylation of alginate.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, P.O. Box 980678, Medical College of Virginia Campus of Virginia Commonwealth University, 1101 E. Marshall St., 5-047 Sanger Hall, Richmond, VA 23298-0678. Phone: (804) 828-9728. Fax: (804) 828-9946. E-mail address: deohman{at}hsc.vcu.edu.

Journal of Bacteriology, June 2002, p. 3000-3007, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3000-3007.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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