Molecular characterization of the major outer-membrane protein OprF from plant root-colonizing Pseudomonas fluorescens.
N-terminal sequence analysis of peptides generated by proteolytic treatment of the Pseudomonas fluorescens OE 28.3 major outer-membrane protein OprF, embedded in outer membranes or present in whole cells, indicated a surface-exposed location for the proline-rich region of the protein. This region is absent from the P. aeruginosa and P. syringae OprFs. Evidence was obtained for the presence of additional exposed but less accessible regions in the carboxy half of OprF. Four OprF-specific monoclonal antibodies were all directed to the C-terminal part of the protein but did not recognize a surface-exposed epitope as shown by flow cytometry. Our data support the model previously proposed for P. aeruginosa OprF in which the entire protein is embedded in the outer membrane, unlike the topology proposed for the major outer-membrane protein from Escherichia coli, OmpA, whose carboxy half resides in the periplasmic space. For six other P. fluorescens strains producing OprF proteins with different isoelectric points, the primary structure was determined by sequence analysis of the PCR-amplified oprF genes. The proline-rich domain represented the most conserved region of the different P. fluorescens OprFs. Based on the sequence of its oprF gene, it was shown that the mushroom pathogen P. tolaasii is quite closely related to P. fluorescens. Comparative sequence analysis further showed that the carboxy half of OprF contains a sequence motif that is well conserved in the enterobacterial OmpA proteins but is also present in a number of other outer-membrane proteins, including peptidoglycan-associated lipoproteins.
De Mot
R
R
F. A. Janssens Laboratory of Genetics, Catholic University of Leuven, Heverlee, Belgium.
Schoofs
G
G
Roelandt
A
A
Declerck
P
P
Proost
P
P
Van Damme
J
J
Vanderleyden
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D001483:Base Sequence; D010450:Endopeptidases / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology; D007526:Isoelectric Point; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010944:Plants / Q000382:microbiology; D016133:Polymerase Chain Reaction; D018272:Porins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011549:Pseudomonas / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D011551:Pseudomonas fluorescens / Q000187:drug effects / Q000235:genetics* / Q000276:immunology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D018272:Porins; D010450:Endopeptidases