Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa
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2002-06
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Alginate is an extracellular polysaccharide produced by mucoid strains of Pseudomonas aeruginosa that are typically isolated from the pulmonary tract 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 positions 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 alginate O acetylation, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar deletions of algI, algJ, and algF were constructed in the FRD1 strain background, 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) fusions were constructed in algF, algJ, and algI. 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 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 fusions were also used to map the membrane topology of AlgI, which 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.
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Franklin MJ, Ohman DE, "Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa," J Bacteriology, 2002 184(11):3000-3007