Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jun;179(12):3884–3891. doi: 10.1128/jb.179.12.3884-3891.1997

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

K K Wallace 1, S Lobo 1, L Han 1, H A McArthur 1, K A Reynolds 1
PMCID: PMC179196  PMID: 9190803

Abstract

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

Full Text

The Full Text of this article is available as a PDF (178.8 KB).

Selected References

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

  1. Arimura N., Kaneda T. Type selective inhibition of microbial fatty acid synthases by thiolactomycin. Arch Microbiol. 1993;160(2):158–161. doi: 10.1007/BF00288719. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Butterworth P. H., Bloch K. Comparative aspects of fatty acid synthesis in Bacillus subtilis and Escherichia coli. Eur J Biochem. 1970 Feb;12(3):496–501. doi: 10.1111/j.1432-1033.1970.tb00878.x. [DOI] [PubMed] [Google Scholar]
  4. Clough R. C., Matthis A. L., Barnum S. R., Jaworski J. G. Purification and characterization of 3-ketoacyl-acyl carrier protein synthase III from spinach. A condensing enzyme utilizing acetyl-coenzyme A to initiate fatty acid synthesis. J Biol Chem. 1992 Oct 15;267(29):20992–20998. [PubMed] [Google Scholar]
  5. Denoya C. D., Fedechko R. W., Hafner E. W., McArthur H. A., Morgenstern M. R., Skinner D. D., Stutzman-Engwall K., Wax R. G., Wernau W. C. A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins. J Bacteriol. 1995 Jun;177(12):3504–3511. doi: 10.1128/jb.177.12.3504-3511.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gulliver B. S., Slabas A. R. Acetoacyl-acyl carrier protein synthase from avocado: its purification, characterisation and clear resolution from acetyl CoA:ACP transacylase. Plant Mol Biol. 1994 May;25(2):179–191. doi: 10.1007/BF00023236. [DOI] [PubMed] [Google Scholar]
  7. Hale R. S., Jordan K. N., Leadlay P. F. A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in Streptomyces erythraeus. FEBS Lett. 1987 Nov 16;224(1):133–136. doi: 10.1016/0014-5793(87)80436-2. [DOI] [PubMed] [Google Scholar]
  8. Hayashi T., Yamamoto O., Sasaki H., Kawaguchi A., Okazaki H. Mechanism of action of the antibiotic thiolactomycin inhibition of fatty acid synthesis of Escherichia coli. Biochem Biophys Res Commun. 1983 Sep 30;115(3):1108–1113. doi: 10.1016/s0006-291x(83)80050-3. [DOI] [PubMed] [Google Scholar]
  9. Hayashi T., Yamamoto O., Sasaki H., Okazaki H., Kawaguchi A. Inhibition of fatty acid synthesis by the antibiotic thiolactomycin. J Antibiot (Tokyo) 1984 Nov;37(11):1456–1461. doi: 10.7164/antibiotics.37.1456. [DOI] [PubMed] [Google Scholar]
  10. Jackowski S., Murphy C. M., Cronan J. E., Jr, Rock C. O. Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin. J Biol Chem. 1989 May 5;264(13):7624–7629. [PubMed] [Google Scholar]
  11. Jackowski S., Rock C. O. Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria. J Biol Chem. 1987 Jun 5;262(16):7927–7931. [PubMed] [Google Scholar]
  12. Kaneda T. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol Rev. 1991 Jun;55(2):288–302. doi: 10.1128/mr.55.2.288-302.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaneda T., Smith E. J. Relationship of primer specificity of fatty acid de novo synthetase to fatty acid composition in 10 species of bacteria and yeasts. Can J Microbiol. 1980 Aug;26(8):893–898. doi: 10.1139/m80-155. [DOI] [PubMed] [Google Scholar]
  14. Magnuson K., Jackowski S., Rock C. O., Cronan J. E., Jr Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev. 1993 Sep;57(3):522–542. doi: 10.1128/mr.57.3.522-542.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nishida I., Kawaguchi A., Yamada M. Effect of thiolactomycin on the individual enzymes of the fatty acid synthase system in Escherichia coli. J Biochem. 1986 May;99(5):1447–1454. doi: 10.1093/oxfordjournals.jbchem.a135614. [DOI] [PubMed] [Google Scholar]
  16. Noto T., Miyakawa S., Oishi H., Endo H., Okazaki H. Thiolactomycin, a new antibiotic. III. In vitro antibacterial activity. J Antibiot (Tokyo) 1982 Apr;35(4):401–410. doi: 10.7164/antibiotics.35.401. [DOI] [PubMed] [Google Scholar]
  17. Oishi H., Noto T., Sasaki H., Suzuki K., Hayashi T., Okazaki H., Ando K., Sawada M. Thiolactomycin, a new antibiotic. I. Taxonomy of the producing organism, fermentation and biological properties. J Antibiot (Tokyo) 1982 Apr;35(4):391–395. doi: 10.7164/antibiotics.35.391. [DOI] [PubMed] [Google Scholar]
  18. Revill W. P., Bibb M. J., Hopwood D. A. Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant. J Bacteriol. 1995 Jul;177(14):3946–3952. doi: 10.1128/jb.177.14.3946-3952.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Revill W. P., Leadlay P. F. Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis. J Bacteriol. 1991 Jul;173(14):4379–4385. doi: 10.1128/jb.173.14.4379-4385.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rezanka T., Reichelová J., Kopecký J. Isobutyrate as a precursor of n-butyrate in the biosynthesis of tylosine and fatty acids. FEMS Microbiol Lett. 1991 Nov 1;68(1):33–36. doi: 10.1016/0378-1097(91)90390-v. [DOI] [PubMed] [Google Scholar]
  21. Rossi A., Corcoran J. W. Identification of a multienzyme complex synthesizing fatty acids in the actinomycete Streptomyces erythreus. Biochem Biophys Res Commun. 1973 Feb 5;50(3):597–602. doi: 10.1016/0006-291x(73)91286-2. [DOI] [PubMed] [Google Scholar]
  22. Saito K., Kawaguchi A., Seyama Y., Yamakawa T., Okuda S. Steric course of deuterium incorporation from [2-2H2]malonyl-CoA into fatty acids by fatty acid synthetases. J Biochem. 1981 Dec;90(6):1697–1704. doi: 10.1093/oxfordjournals.jbchem.a133646. [DOI] [PubMed] [Google Scholar]
  23. Summers R. G., Ali A., Shen B., Wessel W. A., Hutchinson C. R. Malonyl-coenzyme A:acyl carrier protein acyltransferase of Streptomyces glaucescens: a possible link between fatty acid and polyketide biosynthesis. Biochemistry. 1995 Jul 25;34(29):9389–9402. doi: 10.1021/bi00029a015. [DOI] [PubMed] [Google Scholar]
  24. Suutari M., Laakso S. Changes in fatty acid branching and unsaturation of Streptomyces griseus and Brevibacterium fermentans as a response to growth temperature. Appl Environ Microbiol. 1992 Jul;58(7):2338–2340. doi: 10.1128/aem.58.7.2338-2340.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tsay J. T., Oh W., Larson T. J., Jackowski S., Rock C. O. Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12. J Biol Chem. 1992 Apr 5;267(10):6807–6814. [PubMed] [Google Scholar]
  26. Tsay J. T., Rock C. O., Jackowski S. Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12. J Bacteriol. 1992 Jan;174(2):508–513. doi: 10.1128/jb.174.2.508-513.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wallace K. K., Bao Z. Y., Dai H., Digate R., Schuler G., Speedie M. K., Reynolds K. A. Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli. Eur J Biochem. 1995 Nov 1;233(3):954–962. doi: 10.1111/j.1432-1033.1995.954_3.x. [DOI] [PubMed] [Google Scholar]
  28. Wallace K. K., Zhao B., McArthur H. A., Reynolds K. A. In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes. FEMS Microbiol Lett. 1995 Sep 1;131(2):227–234. doi: 10.1111/j.1574-6968.1995.tb07781.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES