Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.
The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes.
Siehnel
R J
RJ
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Worobec
E A
EA
Hancock
R E
RE
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007118:Immunoassay; D009693:Nucleic Acid Hybridization; D018272:Porins; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D018272:Porins; D004262:DNA Restriction Enzymes