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Structure, function, and role in antibiotic resistance of outer membrane protein H1 in Pseudomonas aeruginosa Bell, Angus


A divalent cation-regulated outer membrane protein of Pseudomonas aeruginosa, HI, was purified by selective solubilizations of outer membranes in detergent and ethylenediaminetraacetate (EDTA) followed by either two cycles of anion-exchange chromatography or sodium dodecylsulphate-polyacrylamide gel electrophoresis. Protein purified by the former method was contaminated with an equi-molar or higher concentration of lipopolysaccharide , (LPS) that was enriched in 0 side chain-containing molecules, suggesting an association between protein Hi and smooth LPS. Electrophoresis gave higher yields of purified protein Hi, and this product was used for N-terminal amino acid sequencing, amino acid analysis, and polyclonal antiserum production. Oligodeoxyribonucleotides complementary to the upstream end of the structural gene for protein Hi, oprH, were designed using the N-terminal sequence of the protein. Probing of Southern blots of chromosomal DNA digests with the oligonucleotides revealed that oprH was probably a single-copy gene, and allowed it to be cloned in Escherichia coli. Nucleotide sequence analysis confirmed the cloning of the correct gene, and the derived amino acid sequence indicated a slightly basic protein (in agreement with its proposed function of interacting with anionic sites on LPS) of 178 residues, with two hydrophobic segments. Protein H1 was produced, proteolytically processed and probably exported to the outer membrane in E. coli cells carrying the complete oprH gene on plasmids. The oprH gene could be expressed weakly from a promoter on the cloned DNA provided that a particular downstream sequence was not deleted. This suggested that the downstream region was involved in regulation of expression of the cloned oprH gene. Protein H1 was produced at levels much higher than background when extra copies of the oprH gene were present in an expression vector in P. aeruginosa. This overproduction of protein H1 caused decreased susceptibility of cells to killing by EDTA, but not by polymyxin B or gentamicin. This partly confirmed the hypothesis (T.I. Nicas and R.E.W. Hancock, J. Bacteriol. 143; 872-878, 1980) that protein HI causes resistance to these agents by inhibiting their self-promoted uptake across the outer membrane. However, an additional alteration (possibly an increase in cationic substituents on LPS) was apparently required for the fully resistant phenotype. This idea was supported by the observed suppression by LPS mutations of the polymyxin B resistance of protein H1-overproducing cells, and by the properties of a P. aeruginosa strain apparently lacking protein H1. Several species of bacteria related to P. aeruginosa produced envelope proteins that were inducible by growth in Mg²⁺-deficient medium, and one (a protein of apparent molecular weight 20,000 from P. chloraphis) cross-reacted immunologically with protein Hi. P. chloraphis, like P. aeruginosa, was polymyxin B resistant when it was grown in Mg²⁺-deficient medium.

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