TY - JOUR
T1 - Outer membrane protein H1 of Pseudomonas aeruginosa
T2 - Purification of the protein and cloning and nucleotide sequence of the gene
AU - Bell, A.
AU - Hancock, R. E.W.
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1989
Y1 - 1989
N2 - Overexpression of the divalent cation-regulated outer membrane protein H1 of Pseudominas 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 disruptoin 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 acid 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 revelaed 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 conclude that, as for some other regulated Pseudomonas genes, expression of oprH, at least under some conditions, is blocked in E. coli.
AB - Overexpression of the divalent cation-regulated outer membrane protein H1 of Pseudominas 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 disruptoin 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 acid 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 revelaed 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 conclude that, as for some other regulated Pseudomonas genes, expression of oprH, at least under some conditions, is blocked in E. coli.
UR - http://www.scopus.com/inward/record.url?scp=0024311582&partnerID=8YFLogxK
U2 - 10.1128/jb.171.6.3211-3217.1989
DO - 10.1128/jb.171.6.3211-3217.1989
M3 - Article
C2 - 2498288
AN - SCOPUS:0024311582
SN - 0021-9193
VL - 171
SP - 3211
EP - 3217
JO - Journal of Bacteriology
JF - Journal of Bacteriology
IS - 6
ER -