This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Franklin, M. J. Articles by Ohman, D. E. Search for Related Content PubMed PubMed Citation Articles by Franklin, M. J. Articles by Ohman, D. E.

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

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.

This article has been cited by other articles:

• Gross, C., Felsheim, R., Wackett, L. P. (2008). Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic. J. Bacteriol. 190: 4859-4864 [Abstract] [Full Text]  
• Oglesby, L. L., Jain, S., Ohman, D. E. (2008). Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization. Microbiology 154: 1605-1615 [Abstract] [Full Text]  
• Gimmestad, M., Steigedal, M., Ertesvag, H., Moreno, S., Christensen, B. E., Espin, G., Valla, S. (2006). Identification and Characterization of an Azotobacter vinelandii Type I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases.. J. Bacteriol. 188: 5551-5560 [Abstract] [Full Text]  
• Remminghorst, U., Rehm, B. H. A. (2006). In Vitro Alginate Polymerization and the Functional Role of Alg8 in Alginate Production by Pseudomonas aeruginosa. Appl. Environ. Microbiol. 72: 298-305 [Abstract] [Full Text]  
• Bakkevig, K., Sletta, H., Gimmestad, M., Aune, R., Ertesvag, H., Degnes, K., Christensen, B. E., Ellingsen, T. E., Valla, S. (2005). Role of the Pseudomonas fluorescens Alginate Lyase (AlgL) in Clearing the Periplasm of Alginates Not Exported to the Extracellular Environment. J. Bacteriol. 187: 8375-8384 [Abstract] [Full Text]  
• Jain, S., Ohman, D. E. (2005). Role of an Alginate Lyase for Alginate Transport in Mucoid Pseudomonas aeruginosa. Infect. Immun. 73: 6429-6436 [Abstract] [Full Text]  
• Douthit, S. A., Dlakic, M., Ohman, D. E., Franklin, M. J. (2005). Epimerase Active Domain of Pseudomonas aeruginosa AlgG, a Protein That Contains a Right-Handed {beta}-Helix. J. Bacteriol. 187: 4573-4583 [Abstract] [Full Text]  
• Albrecht, M. T., Schiller, N. L. (2005). Alginate Lyase (AlgL) Activity Is Required for Alginate Biosynthesis in Pseudomonas aeruginosa. J. Bacteriol. 187: 3869-3872 [Abstract] [Full Text]  
• Hoffmann, N., Rasmussen, T. B., Jensen, P., Stub, C., Hentzer, M., Molin, S., Ciofu, O., Givskov, M., Johansen, H. K., Hoiby, N. (2005). Novel Mouse Model of Chronic Pseudomonas aeruginosa Lung Infection Mimicking Cystic Fibrosis. Infect. Immun. 73: 2504-2514 [Abstract] [Full Text]  
• Gudlavalleti, S. K., Datta, A. K., Tzeng, Y.-L., Noble, C., Carlson, R. W., Stephens, D. S. (2004). The Neisseria meningitidis Serogroup A Capsular Polysaccharide O-3 and O-4 Acetyltransferase. J. Biol. Chem. 279: 42765-42773 [Abstract] [Full Text]  
• Caiazza, N. C., O'Toole, G. A. (2004). SadB Is Required for the Transition from Reversible to Irreversible Attachment during Biofilm Formation by Pseudomonas aeruginosa PA14. J. Bacteriol. 186: 4476-4485 [Abstract] [Full Text]  
• Franklin, M. J., Douthit, S. A., McClure, M. A. (2004). Evidence that the algI/algJ Gene Cassette, Required for O Acetylation of Pseudomonas aeruginosa Alginate, Evolved by Lateral Gene Transfer. J. Bacteriol. 186: 4759-4773 [Abstract] [Full Text]  
• Nascimento, A. L. T. O., Ko, A. I., Martins, E. A. L., Monteiro-Vitorello, C. B., Ho, P. L., Haake, D. A., Verjovski-Almeida, S., Hartskeerl, R. A., Marques, M. V., Oliveira, M. C., Menck, C. F. M., Leite, L. C. C., Carrer, H., Coutinho, L. L., Degrave, W. M., Dellagostin, O. A., El-Dorry, H., Ferro, E. S., Ferro, M. I. T., Furlan, L. R., Gamberini, M., Giglioti, E. A., Goes-Neto, A., Goldman, G. H., Goldman, M. H. S., Harakava, R., Jeronimo, S. M. B, Junqueira-de-Azevedo, I. L. M., Kimura, E. T., Kuramae, E. E., Lemos, E. G. M., Lemos, M. V. F., Marino, C. L., Nunes, L. R., de Oliveira, R. C., Pereira, G. G., Reis, M. S., Schriefer, A., Siqueira, W. J., Sommer, P., Tsai, S. M., Simpson, A. J. G., Ferro, J. A., Camargo, L. E. A., Kitajima, J. P., Setubal, J. C., Van Sluys, M. A. (2004). Comparative Genomics of Two Leptospira interrogans Serovars Reveals Novel Insights into Physiology and Pathogenesis. J. Bacteriol. 186: 2164-2172 [Abstract] [Full Text]  
• Neuhaus, F. C., Baddiley, J. (2003). A Continuum of Anionic Charge: Structures and Functions of D-Alanyl-Teichoic Acids in Gram-Positive Bacteria. Microbiol. Mol. Biol. Rev. 67: 686-723 [Abstract] [Full Text]  
• Gimmestad, M., Sletta, H., Ertesvag, H., Bakkevig, K., Jain, S., Suh, S.-j., Skjak-Braek, G., Ellingsen, T. E., Ohman, D. E., Valla, S. (2003). The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation. J. Bacteriol. 185: 3515-3523 [Abstract] [Full Text]