1887

Abstract

Summary: The first characterization of fatty acid uptake in a Gram-positive bacterium is reported. A3(2) utilizes fatty acids of different chain length (C-C) as sole carbon and energy sources. In vivo β-oxidation studies and the assay of two enzymes of the β-oxidation cycle proved that fatty acid degradation is constitutive in this micro-organism. Uptake of the medium-chain fatty acid octanoate showed the characteristics of simple diffusion, whereas the uptake of palmitate, a long-chain fatty acid, occurred by both simple diffusion and active transport. After correcting for non-mediated transport, palmitate uptake measured over a wide range of concentrations followed Michaelis-Menten kinetics. The apparent for palmitate was 97.8 μM and the was 19.3 nmol min (mg protein) Competition experiments showed specificity of the mediated transport component for long-chain fatty acids (> C). Metabolic inhibitors such as oligomycin, NaF and vanadate, and the ionophores gramicidin and carbonyl cyanide -chlorophenylhydrazone (CCCP) inhibited palmitate uptake to different degrees, consistent with the existence of an active transport mechanism. Uptake rates measured at different pH values indicated that both the ionized and the unionized forms of octanoate crossed the cytoplasmic membrane by simple diffusion. Palmitate in its ionized form appears to be transported by an active mechanism, whereas the unionized molecule diffuses through the membrane. When present in the medium, glucose stimulated the degradation of long-chain fatty acids by increasing the rate of uptake and the level of acyl-CoA synthetase.

Funding
This study was supported by the:
  • Fundacion Antorchas, the International Foundation for Science (IFS)
  • National Research Council of Argentina (CONICET)
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1997-07-01
2021-06-18
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References

  1. Black P. N. 1988; The fadL gene product of Escherichia coli: an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriphage T2. J Bacteriol 170:2850–2854
    [Google Scholar]
  2. Black P. N., DiRusso C. 1994; Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation by Escherichia coli . Biochim Biophys Acta 1210:123–145
    [Google Scholar]
  3. Black P. N., Klanian S. F., DiRusso C. C., Nunn W. D. 1985; Long chain fatty acid transport in Escherichia coli: cloning, mapping and expression of the fadL gene. J Biol Chem 260:1780–1790
    [Google Scholar]
  4. Clark D., Cronan J. E. , Jr 1996 Two-carbon compounds and fatty acids as carbon sources. In Escherichia coli and Salmonella: Cellular and Molecular Biology , pp 343–357 Edited by Neidhardt F. C. and others Washington, DC: American Society for Microbiology.;
    [Google Scholar]
  5. Delic I., Robbins P., Westpheling J. 1992; Direct repeat sequences are implicated in the regulation of two Streptomyces chitinase promoters that are subject to carbon catabolite control. Proc Natl Acad Sci USA 89:1885–1889
    [Google Scholar]
  6. Fornwald J. A., Schmidt R. J., Adams C. W., Rosenberg M., Brawner M. E. 1987; Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon. Proc Natl Acad Sci USA 84:2130–2134
    [Google Scholar]
  7. Ginsburgh C. L., Black P. N., Nunn W. D. 1984; Transport of long chain fatty acids in Escherichia coli. Identification of a membrane protein associated with the fadL gene. J Biol Chem 259:8437–8443
    [Google Scholar]
  8. Hamilton J. A., Cistola D. A. 1986; Transfer of oleic acid between albumin and phospholipid vesicles. Proc Natl Acad Sci USA 83:82–86
    [Google Scholar]
  9. Hodgson D. 1982; Glucose repression of carbon source uptake in Streptomyces coelicolor A3 (2) and its perturbation in mutants resistant to 2-deoxyglucose. J Gen Microbiol 128:2417–2430
    [Google Scholar]
  10. Hopwood D. A., Kieser T., Wright H. M., Bibb M. J. 1983; Plasmids, recombination and chromosome mapping in Streptomyces lividans 66 . J Gen Microbiol 129:2257–2269
    [Google Scholar]
  11. Hopwood D. A., Bibb M. J., Chater K. F., Kieser, T” Bruton C. J., Kieser H. M., Lydiate M. J., Smith C. P., Ward J. M., Schrempf H. 1985 Genetic Manipulation of Streptomyces: a Laboratory Manual Norwich: John Innes Foundation;
    [Google Scholar]
  12. Hopwood D. A., Chater K. F., Bibb M. J. 1994 Genetics of antibiotic production in Streptomyces coelicolor A3 (2), a model streptomycete. . In Genetics and Biochemistry of Antibiotic Production , pp. 65–102 . Edited by Vining L. C., Stuttard C. Philadelphia: Butterworth-Heinemann;
    [Google Scholar]
  13. Kamp F., Hamilton J. 1992; pH gradients across phospholipid membranes caused by fast flip-flop of unionized fatty acids. Proc Natl Acad Sci USA 89:11367–11370
    [Google Scholar]
  14. Klein K., Steinberg R., Fietchen B., Overath P. 1971; Fatty acid degradation in Escherichia coli. An inducible system for the uptake of fatty acids and further characterization of oW-mutants. Eur } Biochem 19:4281–4290
    [Google Scholar]
  15. Korn-Wendisch F., Kutzner H. J. 1991 The family Strepto- mycetaceae. . In The Prokaryotes , pp. 921–995 Edited by Triiper A. H., Dworkin M., Harder W., Schleifer K. New York: Springer;
    [Google Scholar]
  16. Miyashita K., Fujii T., Sawada Y. 1991; Molecular cloning and characterisation of chitinase genes from Streptomyces lividans 66. Gene 137:2065–2072
    [Google Scholar]
  17. Nunn W. D. 1986; A molecular view of fatty acid catabolism in Escherichia coli . Microbiol Rev 50:179–192
    [Google Scholar]
  18. Nunn W. D., Simons R. W., Egan P. A., Maloy S. R. 1979; Kinetics of the utilization of medium and long-chain fatty acid degradation by a mutant of Escherichia coli defective in the fadL gene. J Biol Chem 254:9130–9134
    [Google Scholar]
  19. Olukoshi E. R., Packter N. M. 1994; Importance of stored triacylglycerols in Streptomyces: possible carbon source for antibiotics. Microbiology 140:93–943
    [Google Scholar]
  20. Overath P., Raufuss E. M., Stoffel W., Ecker W. 1967; The induction of the enzymes of fatty acid degradation of Escherichia coli . Biochem Biophys Res Commun 29:28–33
    [Google Scholar]
  21. Overath P., Pauli G., Schairer H. U. 1969; Fatty acid degradation in Escherichia coli. An inducible acyl-CoA synthetase, the mapping of old-mutations, and the isolation of regulatory mutants. Eur J Biochem 7:559–574
    [Google Scholar]
  22. Pauli G., Erhing R., & Overath P. 1974; Fatty acid degradation in Escherichia coli: requirement of cyclic adenosine monophosphate and cyclic adenosine monophosphate receptor protein for enzyme synthesis. J Bacteriol 117:1178–1183
    [Google Scholar]
  23. Peczynska-Czoch W., Mordarski M. 1988 Actinomycete enzymes. . In Actinomycetes in Biotechnology , pp. 220-283. Edited by Goodfellow M., Williams S. T., Mordarski M. San Diego, CA: Academic Press;
    [Google Scholar]
  24. Pérez G,, Juárez K., Garcfa-Castells E., Sober6n G., Servfn- Gonzalez L. 1993; Cloning, characterization, and expression in Streptomyces lividans 66 of an extracellular lipase-encoding gene from Streptomyces sp. Mil. Gene 123:109–114
    [Google Scholar]
  25. Robbins P. W., Overbye K., Albright C., Benfield B., Pero J. 1992; Cloning and high-level expression of a chitinase-encoding gene of Streptomyces plicatus . Gene 111:69–76
    [Google Scholar]
  26. Sato S., Imamura S., Ozeki Y., Kawaguchi A. 1992; Induction of enzymes involved in fatty acid beta-oxidation in Pseudomonas fragi B-0771 cells grown in media supplemented with fatty acid. J Biochem 111:16–19
    [Google Scholar]
  27. Servín-González L., Bibb M. J. 1994; Transcriptional regulation of the four promoters of the agarase gene (dagA) of Streptomyces coelicolor A3(2). Microbiology 140:2555–2565
    [Google Scholar]
  28. Simons R. W., Egan P. A., Crute H. T., Nunn W. D. 1980; Regulation of fatty acid degradation in Escherichia coli: isolation and characterization of strains bearing insertion and temperature- sensitive mutations in gene fadR . J Bacteriol 142:621–632
    [Google Scholar]
  29. Smith C. P., Chater K. F. 1988; Structure and controlling sequences for the Streptomyces coelicolor glycerol operon. J Mol Biol 204:569–580
    [Google Scholar]
  30. Stackebrandt E., Liesack W., Witt D. 1992; Ribosomal RNA and rDNA sequence analysis. Gene 115:255–260
    [Google Scholar]
  31. Tanaka T., Hosaka K., Numa S. 1979; Purification and properties of long-chain acyl-coenzyme-A synthetase from rat liver. Eur J Biochem 98:165–172
    [Google Scholar]
  32. Takano E., Gramajo H. C., Strauch E., Andres N., Bibb M. J. 1992; Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Mol Microbiol 6:2797–2804
    [Google Scholar]
  33. Virolle M. J., Bibb M. J. 1988; Cloning, characterization and regulation of an α-amylase gene from Streptomyces limosus . Mol Microbiol 2:197–208
    [Google Scholar]
  34. Weast R. C. 1973 Handbook of Chemistry and Physics, D-129. Cleveland, OH: CRC Press;
    [Google Scholar]
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