Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Oct;178(20):6025–6035. doi: 10.1128/jb.178.20.6025-6035.1996

Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA.

R G Kok 1, C B Nudel 1, R H Gonzalez 1, I M Nugteren-Roodzant 1, K J Hellingwerf 1
PMCID: PMC178462  PMID: 8830702

Abstract

The extracellular lipase (LipA) produced by Acinetobacter calcoaceticus BD413 is required for growth of the organism on triolein, since mutant strains that lack an active lipase fail to grow with triolein as the sole carbon source. Surprisingly, extracellular lipase activity and expression of the structural lipase gene (lipA), the latter measured through lacZ as a transcriptional reporter, are extremely low in triolein cultures of LipA+ strains. The explanation for this interesting paradox lies in the effect of fatty acids on the expression of lipA. We found that long-chain fatty acids, especially, strongly repress the expression of lipA, thereby negatively influencing the production of lipase. We propose the involvement of a fatty acyl-responsive DNA-binding protein in regulation of expression of the A. calcoaceticus lipBA operon. The potential biological significance of the observed physiological competition between expression and repression of lipA in the triolein medium is discussed. Activity of the extracellular lipase is also negatively affected by proteolytic degradation, as shown in in vitro stability experiments and by Western blotting (immunoblotting) of concentrated supernatants of stationary-phase cultures. In fact, the relatively high levels of extracellular lipase produced in the early stationary phase in media which contain hexadecane are due only to enhanced stability of the extracellular enzyme under those conditions. The rapid extracellular degradation of LipA of A. calcoaceticus BD413 by an endogenous protease is remarkable and suggests that proteolytic degradation of the enzyme is another important factor in regulating the level of active extracellular lipase.

Full Text

The Full Text of this article is available as a PDF (480.9 KB).

Selected References

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

  1. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. DiRusso C. C., Metzger A. K., Heimert T. L. Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichia coli by FadR. Mol Microbiol. 1993 Jan;7(2):311–322. doi: 10.1111/j.1365-2958.1993.tb01122.x. [DOI] [PubMed] [Google Scholar]
  3. Frenken L. G., Bos J. W., Visser C., Müller W., Tommassen J., Verrips C. T. An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase. Mol Microbiol. 1993 Aug;9(3):579–589. doi: 10.1111/j.1365-2958.1993.tb01718.x. [DOI] [PubMed] [Google Scholar]
  4. Frenken L. G., Egmond M. R., Batenburg A. M., Bos J. W., Visser C., Verrips C. T. Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues. Appl Environ Microbiol. 1992 Dec;58(12):3787–3791. doi: 10.1128/aem.58.12.3787-3791.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Frenken L. G., Egmond M. R., Batenburg A. M., Verrips C. T. Pseudomonas glumae lipase: increased proteolytic stability by protein engineering. Protein Eng. 1993 Aug;6(6):637–642. doi: 10.1093/protein/6.6.637. [DOI] [PubMed] [Google Scholar]
  6. Frenken L. G., de Groot A., Tommassen J., Verrips C. T. Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae. Mol Microbiol. 1993 Aug;9(3):591–599. doi: 10.1111/j.1365-2958.1993.tb01719.x. [DOI] [PubMed] [Google Scholar]
  7. Gilbert E. J., Drozd J. W., Jones C. W. Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2. J Gen Microbiol. 1991 Sep;137(9):2215–2221. doi: 10.1099/00221287-137-9-2215. [DOI] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  9. Henry M. F., Cronan J. E., Jr A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli. J Bacteriol. 1989 Oct;171(10):5254–5261. doi: 10.1128/jb.171.10.5254-5261.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henry M. F., Cronan J. E., Jr A new mechanism of transcriptional regulation: release of an activator triggered by small molecule binding. Cell. 1992 Aug 21;70(4):671–679. doi: 10.1016/0092-8674(92)90435-f. [DOI] [PubMed] [Google Scholar]
  11. Hobson A. H., Buckley C. M., Aamand J. L., Jørgensen S. T., Diderichsen B., McConnell D. J. Activation of a bacterial lipase by its chaperone. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5682–5686. doi: 10.1073/pnas.90.12.5682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Iizumi T., Nakamura K., Shimada Y., Sugihara A., Tominaga Y., Fukase T. Cloning, nucleotide sequencing, and expression in Escherichia coli of a lipase and its activator genes from Pseudomonas sp. KWI-56. Agric Biol Chem. 1991 Sep;55(9):2349–2357. [PubMed] [Google Scholar]
  13. Jaeger K. E., Ransac S., Dijkstra B. W., Colson C., van Heuvel M., Misset O. Bacterial lipases. FEMS Microbiol Rev. 1994 Sep;15(1):29–63. doi: 10.1111/j.1574-6976.1994.tb00121.x. [DOI] [PubMed] [Google Scholar]
  14. Juni E. Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J Bacteriol. 1972 Nov;112(2):917–931. doi: 10.1128/jb.112.2.917-931.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Juni E. Simple genetic transformation assay for rapid diagnosis of Moraxella osloensis. Appl Microbiol. 1974 Jan;27(1):16–24. doi: 10.1128/am.27.1.16-24.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jørgensen S., Skov K. W., Diderichsen B. Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes. J Bacteriol. 1991 Jan;173(2):559–567. doi: 10.1128/jb.173.2.559-567.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kok R. G., Christoffels V. M., Vosman B., Hellingwerf K. J. Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases. J Gen Microbiol. 1993 Oct;139(10):2329–2342. doi: 10.1099/00221287-139-10-2329. [DOI] [PubMed] [Google Scholar]
  18. Kok R. G., van Thor J. J., Nugteren-Roodzant I. M., Brouwer M. B., Egmond M. R., Nudel C. B., Vosman B., Hellingwerf K. J. Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene. Mol Microbiol. 1995 Mar;15(5):803–818. doi: 10.1111/j.1365-2958.1995.tb02351.x. [DOI] [PubMed] [Google Scholar]
  19. Kok R. G., van Thor J. J., Nugteren-Roodzant I. M., Vosman B., Hellingwerf K. J. Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase. J Bacteriol. 1995 Jun;177(11):3295–3307. doi: 10.1128/jb.177.11.3295-3307.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Landt O., Grunert H. P., Hahn U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990 Nov 30;96(1):125–128. doi: 10.1016/0378-1119(90)90351-q. [DOI] [PubMed] [Google Scholar]
  22. Nunn W. D. A molecular view of fatty acid catabolism in Escherichia coli. Microbiol Rev. 1986 Jun;50(2):179–192. doi: 10.1128/mr.50.2.179-192.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Palmen R., Vosman B., Buijsman P., Breek C. K., Hellingwerf K. J. Physiological characterization of natural transformation in Acinetobacter calcoaceticus. J Gen Microbiol. 1993 Feb;139(2):295–305. doi: 10.1099/00221287-139-2-295. [DOI] [PubMed] [Google Scholar]
  24. Quail M. A., Dempsey C. E., Guest J. R. Identification of a fatty acyl responsive regulator (FarR) in Escherichia coli. FEBS Lett. 1994 Dec 19;356(2-3):183–187. doi: 10.1016/0014-5793(94)01264-4. [DOI] [PubMed] [Google Scholar]
  25. Stuer W., Jaeger K. E., Winkler U. K. Purification of extracellular lipase from Pseudomonas aeruginosa. J Bacteriol. 1986 Dec;168(3):1070–1074. doi: 10.1128/jb.168.3.1070-1074.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vosman B., Rauch P. J., Westerhoff H. V., Hellingwerf K. J. Regulation of the expression of the Pseudomonas stutzeri recA gene. Antonie Van Leeuwenhoek. 1993 Jan;63(1):55–62. doi: 10.1007/BF00871732. [DOI] [PubMed] [Google Scholar]

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

RESOURCES