Outer membrane protein H1 of Pseudomonas aeruginosa: purification of the protein and cloning and nucleotide sequence of the gene.
Overexpression of the divalent cation-regulated outer membrane protein H1 of Pseudomonas aeruginosa is associated with resistance to polymyxin B, aminoglycosides, and EDTA. Protein H1 is believed to act by replacing divalent cations at binding sites on lipopolysaccharide, thereby preventing disruption of the sites and subsequent self-promoted uptake of the antibiotics. Protein H1 purified by two cycles of anion-exchange chromatography was apparently associated with lipopolysaccharide. Lipopolysaccharide-free protein H1 was purified in high yield by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was subjected to N-terminal amino sequencing. Complementary oligodeoxyribonucleotides were used to clone the structural gene for protein H1, oprH, into Escherichia coli. Successful cloning was confirmed by nucleotide sequence analysis. Southern hybridization suggested that oprH was present as a single-copy gene in P. aeruginosa. The deduced amino acid sequence revealed that H1 was a slightly basic polypeptide of 178 residues, with a leader sequence typical of an exported procaryotic protein. It had little similarity, however, to other bacterial surface proteins for which sequence data were available. No expression of protein H1, from its own or the lac promoter, was detected in E. coli. We concluded that, as for some other regulated Pseudomonas genes, expression of oprH, at least under some conditions, is blocked in E. coli.
Bell
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Department of Microbiology, University of British Columbia, Vancouver, Canada.
Hancock
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D011550:Pseudomonas aeruginosa / Q000032:analysis / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D015345:Oligonucleotide Probes