Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli

Abstract

The fadL gene of Escherichia coli codes for an outer membrane protein that is involved in the uptake of long-chain fatty acids. Uptake is regulated by environmental osmolarity, and decreases when the cells are grown under conditions of high osmolarity. A temperature-sensitive mutant that requires fatty acid for growth at 42° C was unable to grow at the high temperature even in the presence of fatty acid if the medium contained 10% sucrose. Promoter activity of the fadL gene in vivo was repressed by high osmolarity in a FadR repressor null mutant. Furthermore, in vitro transcription of the fadL gene was strongly repressed by the addition of OmpR and EnvZ proteins. The results of gel retardation and DNase I protection experiments indicated that OmpR, after incubation with the protein kinase EnvZ, specifically binds to at least four sites around the fadL promoter, two upstream and two downstream from the transcriptional start site. These results suggest that transcription of the fadL gene is osmotically regulated by the OmpREnvZ two-component system.

This is a preview of subscription content, log in to check access.

References

  1. Aiba H, Mizuno T (1990) Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, stimulates the transcription of the ompF and ompC genes in Escherichia coli. FEBS Lett 261: 19–22

  2. Aiba H, Nakasai F, Mizushima S, Mizuno T (1989a) Evidence for the physiological importance of the phosphotransfer between the two regulatory components, EnvZ and OmpR, in osmoregulation in Escherichia coli. J Biol Chem 264:14090–14094

  3. Aiba H, Nakasai F, Mizushima S, Mizuno T (1989b) Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability. J Biochem 106:5–7

  4. Aiba H, Mizuno T, Mizushima S (1989c) Transfer of phosphoryl group between two regulatory proteins involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli. J Biol Chem 264:8563–8567

  5. Black PN (1988) The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2. J Bacteriol 170:2850–2854

  6. Black PN (1990) Characterization of FadL-specific fatty acid binding in Escherichia coli. Biochim Biophys Acta 1046:97–105

  7. Black PN (1991) Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport. J Bacteriol 173:435–442

  8. Black PN, Kianian SF, DiRusso CC, Nunn WD (1985) Long-chain fatty acid transport in Escherichia coli: cloning, mapping and expression of the fadL gene. J Biol Chem 260:1780–1789

  9. Black PN, Said B, Ghosn CR, Beach JV, Nunn WD (1987) Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli. J Biol Chem 262:1412–1419

  10. Broekman JHFF, Steenbakkers JF (1974) Effect of the osmotic pressure of the growth medium on fabB mutants of Escherichia coli. J Bacteriol 117:971–977

  11. Brosius J, Lupski JR (1987) Plasmids for the selection and analysis of prokaryotic promoters. Methods Enzymol 153:54–68

  12. Clark L, Carbon J (1976) A colony bank containing synthetic co1E1 hybrid plasmids representative of the entire E. coli genome. Cell 9:91–99

  13. Collado-Vides J, Magasanik B, Gralla JD (1991) Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev 55:371–394

  14. DiRusso CC, Heimert TL, Metzger AK (1992) Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. J Biol Chem 267:8685–8691

  15. Forst S, Delgado J, Rampersaud A, Inouye M (1990) In vivo phosphorylation of OmpR, the transcription activator of the ompF and ompC genes in Escherichia coli. J Bacteriol 172:3473–3477

  16. Galas D, Schmitz A (1978) DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res 5:3157–3170

  17. Gorman CM, Moffat LF, Howard BH (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044–1051

  18. Hall MN, Silhavy TJ (1979) Transcriptional regulation of Escherichia coli K-12 major outer membrane protein lb. J Bacteriol 140:342–350

  19. Hall MN, Silhavy TJ (1981) Genetic analysis of the ompB locus in Escherichia coli K-12. J Mol Biol 151:1–15

  20. Igo MM, Silhavy TJ (1988) EnvZ, a transmembrane environmental sensor of Escherichia coli K-12 is phosphorylated in vitro. J Bacteriol 170:5971–5973

  21. Igo MM, Ninfa AJ, Silhavy TJ (1989a) A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation. Genes Dev 3:598–605

  22. Igo MM, Ninfa AJ, Stock JB, Silhavy TJ (1989b) Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor. Genes Dev 3:1725–1734

  23. Jo Y-L, Nara F, Ichihara S, Mizuno T, Mizushima S (1986) Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli. J Biol Chem 261:15252–15256

  24. Kawaji H, Mizuno T, Mizushima S (1979) Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins O-8 and O-9 of Escherichia coli K12. J Bacteriol 140:843–847

  25. Maeda S, Ozawa Y, Mizuno T, Mizushima S (1988) Stereospecific positioning of the cis-acting sequence with respect to the canonical promoter is required for activation of the ompC gene by a positive regulator, OmpR, in Escherichia coli. J Mol Biol 202:433–441

  26. Maloy SR, Ginsburgh CL, Simons RW, Nunn WD (1981) Transport of long and medium chain fatty acids by Escherichia coli K12. J Biol Chem 256:3735–3742

  27. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  28. Norioka S, Ramakrishnan G, Ikenaka K, Inouye M (1986) Interaction of a transcriptional activator, OmpR, with reciprocally osmoregulated genes, ompF and ompC, of Escherichia coli. J Biol Chem 261:17113–17119

  29. Nunn WD, Simons RW (1978) Transport of long-chain fatty acids by Escherichia coli: mapping and characterization of mutants in the fadL gene. Proc Natl Acad Sci USA 75:3377–3381

  30. Nunn WD, Simons RW, Egan PA, Maloy SR (1979) Kinetics of the utilization of medium- and long-chain fatty acids by a mutant of Escherichia coli defective in the fadL gene. J Biol Chem254:9130–9134

  31. Nunn WD, Colburn RW, Black PN (1986) Transport of long-chain fatty acids in Escherichia coli: evidence for role of fadL gene product as long-chain fatty acid receptor. J Biol Chem 261:167–171

  32. Rampersaud A, Norioka S, Inouye M (1989) Characterization of OmpR binding sequences in the upstream region of the ompF promoter essential for transcriptional activation. J Biol Chem 264:18693–18700

  33. Russo FD, Silhavy TJ (1991) EnvZ controls the concentration of phosphorylated OmpR to mediate osmoregulation of the porin genes. J Mol Biol 222:567–580

  34. Said B, Ghosn CR, Vu L, Nunn WD (1988) Nucleotide sequencing and expression of the fadL gene involved in long-chain fatty acid transport in Escherichia coli. Mol Microbiol 2:363–370

  35. Saier MH Jr, Wu LF, Reizer J (1990) Regulation of bacterial physiological processes by three types of protein phosphorylating systems. Trends Biochem Sci 15:391–395

  36. Sallus L, Haselbeck RJ, Nunn WD (1983) Regulation of fatty acid transport in Escherichia coli: analysis by operon fusion. J Bacteriol 155:1450–1454

  37. Simons RW, Egan PA, Chute HT, Nunn WD (1980a) Regulation of fatty acid degradation in Escherichia coli: isolation and characterization of strains bearing insertion and temperaturesensitive mutations in gene fadR. J Bacteriol 142:621–632

  38. Simons RW, Hughes KT, Nunn WD (1980b) Regulation of fatty acid degradation in Escherichia coli: dominance studies with strains merodiploid in gene fadR. J Bacteriol 143:726–730

  39. Slauch JM, Silhavy TJ (1989) Genetic analysis of the switch that controls porin gene expression in Escherichia coli K-12. J Mol Biol 210:281–292

  40. Slauch JM, Silhavy TJ (1991) cis-Acting ompF mutations that result in OmpR-dependent constitutive expression. J Bacteriol 173:4039–4048

  41. Stock JB, Ninfa AJ, Stock AM (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53:450–490

  42. Suzuki H, Iino T (1973) In vitro synthesis of phase-specific flagellin of Salmonella. J Mol Biol 81:57–70

Download references

Author information

Correspondence to Hirofumi Aiba.

Additional information

Communicated by W. Arber

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Higashitani, A., Nishimura, Y., Hara, H. et al. Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli . Molec. Gen. Genet. 240, 339–347 (1993). https://doi.org/10.1007/BF00280384

Download citation

Key words

  • OmpR
  • EnvZ
  • Phosphorylation
  • Transcriptional control
  • DNA binding