Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Jan;174(2):471–476. doi: 10.1128/jb.174.2.471-476.1992

Overexpression in Escherichia coli and functional analysis of a novel PPi-selective porin, oprO, from Pseudomonas aeruginosa.

R E Hancock 1, C Egli 1, R Benz 1, R J Siehnel 1
PMCID: PMC205739  PMID: 1370289

Abstract

Immediately upstream from and adjacent to the oprP gene, which codes for the phosphate-specific porin OprP of Pseudomonas aeruginosa, lies the PR region (oprO), which cross-hybridizes with oprP DNA. To determine the function of this region, the oprO gene was expressed behind the lactose promoter in Escherichia coli, and the resultant OprO protein was purified and reconstituted into planar lipid bilayers. OprO formed sodium dodecyl sulfate-stable trimers, cross-reacted immunologically with OprP, and, like OprP, formed an anion-specific, phosphate-selective porin. However, it demonstrated lower affinity for and higher maximal conductance of both chloride and phosphate than did the OprP channel. Examination by macroscopic conductance inhibition experiments of the affinity of OprO for phosphates of different lengths revealed a preference for PPi and tripolyphosphate over Pi, suggesting that OprO functioned as a PPi-selective polyphosphate channel, in contrast to OprP, which has a marked preference for Pi.

Full text

PDF
471

Images in this article

472Image
on p.472
473Image
on p.473

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benz R., Darveau R. P., Hancock R. E. Outer-membrane protein PhoE from Escherichia coli forms anion-selective pores in lipid-bilayer membranes. Eur J Biochem. 1984 Apr 16;140(2):319–324. doi: 10.1111/j.1432-1033.1984.tb08104.x. [DOI] [PubMed] [Google Scholar]
  2. Benz R., Hancock R. E. Mechanism of ion transport through the anion-selective channel of the Pseudomonas aeruginosa outer membrane. J Gen Physiol. 1987 Feb;89(2):275–295. doi: 10.1085/jgp.89.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benz R., Schmid A., Hancock R. E. Ion selectivity of gram-negative bacterial porins. J Bacteriol. 1985 May;162(2):722–727. doi: 10.1128/jb.162.2.722-727.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benz R., Schmid A., Nakae T., Vos-Scheperkeuter G. H. Pore formation by LamB of Escherichia coli in lipid bilayer membranes. J Bacteriol. 1986 Mar;165(3):978–986. doi: 10.1128/jb.165.3.978-986.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blasband A. J., Marcotte W. R., Jr, Schnaitman C. A. Structure of the lc and nmpC outer membrane porin protein genes of lambdoid bacteriophage. J Biol Chem. 1986 Sep 25;261(27):12723–12732. [PubMed] [Google Scholar]
  6. Filloux A., Bally M., Soscia C., Murgier M., Lazdunski A. Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of phoB- and phoR-like genes. Mol Gen Genet. 1988 Jun;212(3):510–513. doi: 10.1007/BF00330857. [DOI] [PubMed] [Google Scholar]
  7. Hancock R. E., Benz R. Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa. Biochim Biophys Acta. 1986 Sep 11;860(3):699–707. doi: 10.1016/0005-2736(86)90569-9. [DOI] [PubMed] [Google Scholar]
  8. Hancock R. E., Carey A. M. Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins. J Bacteriol. 1979 Dec;140(3):902–910. doi: 10.1128/jb.140.3.902-910.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Misra R., Benson S. A. A novel mutation, cog, which results in production of a new porin protein (OmpG) of Escherichia coli K-12. J Bacteriol. 1989 Aug;171(8):4105–4111. doi: 10.1128/jb.171.8.4105-4111.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Poole K., Hancock R. E. Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa. Mol Gen Genet. 1986 Mar;202(3):403–409. doi: 10.1007/BF00333269. [DOI] [PubMed] [Google Scholar]
  11. Poole K., Hancock R. E. Phosphate-starvation-induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross-reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa. J Bacteriol. 1986 Mar;165(3):987–993. doi: 10.1128/jb.165.3.987-993.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Siehnel R. J., Worobec E. A., Hancock R. E. Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology. J Bacteriol. 1988 May;170(5):2312–2318. doi: 10.1128/jb.170.5.2312-2318.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Siehnel R. J., Worobec E. A., Hancock R. E. Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coli. Mol Microbiol. 1988 May;2(3):347–352. doi: 10.1111/j.1365-2958.1988.tb00038.x. [DOI] [PubMed] [Google Scholar]
  14. Siehnel R., Martin N. L., Hancock R. E. Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate-specific porin P. Mol Microbiol. 1990 May;4(5):831–838. doi: 10.1111/j.1365-2958.1990.tb00653.x. [DOI] [PubMed] [Google Scholar]
  15. Verhoef C., Benz R., Poon A. P., Tommassen J. New pore protein produced in cells lysogenic for Escherichia coli phage HK253hrk. Eur J Biochem. 1987 Apr 1;164(1):141–145. doi: 10.1111/j.1432-1033.1987.tb11005.x. [DOI] [PubMed] [Google Scholar]
  16. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  17. van der Ley P., Amesz H., Tommassen J., Lugtenberg B. Monoclonal antibodies directed against the cell-surface-exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane. Eur J Biochem. 1985 Mar 1;147(2):401–407. doi: 10.1111/j.1432-1033.1985.tb08764.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES