Polyphosphate-selective porin OprO of Pseudomonas aeruginosa: expression, purification and sequence.
The oprO gene of Pseudomonas aeruginosa codes for a polyphosphate-specific porin and terminates 458 bp upstream of the start codon for the phosphate-specific porin OprP. OprO was found to be expressed only under phosphate-starvation conditions in both wild-type and oprP::Tn501 mutant P. aeruginosa strains. However, unlike the rest of the genes of the Pho regulon, including oprP, expression of oprO required cells to be in the stationary growth phase in addition to phosphate starvation. Wild-type P. aeruginosa cells were grown in fermentor culture under these conditions and fractionated by selective solubilization in octylpolyoxyethylene detergent solution. Solubilized OprO was separated from OprP by application to a Mono Q FPLC column and elution with a salt gradient and shown to be functionally identical to cloned OprO produced in Escherichia coli. DNA sequencing of oprO showed the gene product to be highly homologous to OprP, with 76% identity and 16% conserved substitutions. Most genes of the Pho regulon possess a modified -35 region called the Pho box. Two such elements, separated by 4 bp were found in oprO. DNA sequencing also revealed a second Pho box in the oprP gene with the same spacing.
Siehnel
R J
RJ
Department of Microbiology, University of British Columbia, Vancouver, Canada.
Egli
C
C
Hancock
R E
RE
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D018272:Porins; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017385:Sequence Homology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C072174:OprO protein, Pseudomonas aeruginosa; D018272:Porins; D011994:Recombinant Proteins