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Journal of Bacteriology, September 2008, p. 6119-6125, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00603-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Inner Membrane Retention Signals in Pseudomonas aeruginosa Lipoproteins{triangledown}

Shawn Lewenza,1,3* Musa M. Mhlanga,2 and Anthony P. Pugsley3

University of Calgary, Department of Microbiology & Infectious Diseases, Calgary, Canada,1 Unité de Biologie Cellulaire du Noyau, Institut Pasteur, Paris, France,2 Unité de Génétique Moléculaire, CNRS URA2172, Institut Pasteur, Paris, France3

Received 30 April 2008/ Accepted 7 July 2008

The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFPOmlA-ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFPOmlA-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in CSFPOmlA-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp+2 and Lys+3 Ser+4 function as inner membrane retention signals and identified four novel inner membrane retention signals: CK+2 V+3 E+4, CG+2 G+3 G+4, CG+2 D+3 D+4, and CQ+2 G+3 S+4. These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370CGDD-ChFP and PA3262CDSQ-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.

* Corresponding author. Mailing address: University of Calgary, Department of Microbiology & Infectious Diseases, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1. Phone: (403) 210-7980. Fax: (403) 270-2772. E-mail: slewenza{at}ucalgary.ca

{triangledown} Published ahead of print on 18 July 2008.

Journal of Bacteriology, September 2008, p. 6119-6125, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00603-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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