Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1990 Dec;56(12):3760–3765. doi: 10.1128/aem.56.12.3760-3765.1990

Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli.

P R Contag 1, M G Williams 1, P Rogers 1
PMCID: PMC185064  PMID: 2082823

Abstract

A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.

Full text

PDF
3760

Images in this article

3764Image
on p.3764
3764Image
on p.3764

Selected References

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

  1. Bahl H., Gottwald M., Kuhn A., Rale V., Andersch W., Gottschalk G. Nutritional Factors Affecting the Ratio of Solvents Produced by Clostridium acetobutylicum. Appl Environ Microbiol. 1986 Jul;52(1):169–172. doi: 10.1128/aem.52.1.169-172.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bochner B. R., Savageau M. A. Generalized indicator plate for genetic, metabolic, and taxonomic studies with microorganisms. Appl Environ Microbiol. 1977 Feb;33(2):434–444. doi: 10.1128/aem.33.2.434-444.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  5. Clark D. P. The fermentation pathways of Escherichia coli. FEMS Microbiol Rev. 1989 Sep;5(3):223–234. doi: 10.1016/0168-6445(89)90033-8. [DOI] [PubMed] [Google Scholar]
  6. Cunningham P. R., Clark D. P. The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation. Mol Gen Genet. 1986 Dec;205(3):487–493. doi: 10.1007/BF00338087. [DOI] [PubMed] [Google Scholar]
  7. Davies R., Stephenson M. Studies on the acetone-butyl alcohol fermentation: Nutritional and other factors involved in the preparation of active suspensions of Cl. acetobutylicum (Weizmann). Biochem J. 1941 Dec;35(12):1320–1331. doi: 10.1042/bj0351320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feltham R. K., Power A. K., Pell P. A., Sneath P. A. A simple method for storage of bacteria at--76 degrees C. J Appl Bacteriol. 1978 Apr;44(2):313–316. doi: 10.1111/j.1365-2672.1978.tb00804.x. [DOI] [PubMed] [Google Scholar]
  9. Garvie E. I. Bacterial lactate dehydrogenases. Microbiol Rev. 1980 Mar;44(1):106–139. doi: 10.1128/mr.44.1.106-139.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones D. T., Woods D. R. Acetone-butanol fermentation revisited. Microbiol Rev. 1986 Dec;50(4):484–524. doi: 10.1128/mr.50.4.484-524.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Mat-Jan F., Alam K. Y., Clark D. P. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase. J Bacteriol. 1989 Jan;171(1):342–348. doi: 10.1128/jb.171.1.342-348.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. NOVICK R. P., MAAS W. K. Control by endogenously synthesized arginine of the formation of ornithine transcarbamylase in Escherichia coli. J Bacteriol. 1961 Feb;81:236–240. doi: 10.1128/jb.81.2.236-240.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. O'Brien R. W., Morris J. G. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol. 1971 Nov;68(3):307–318. doi: 10.1099/00221287-68-3-307. [DOI] [PubMed] [Google Scholar]
  15. Palosaari N. R., Rogers P. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum. J Bacteriol. 1988 Jul;170(7):2971–2976. doi: 10.1128/jb.170.7.2971-2976.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  17. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tarmy E. M., Kaplan N. O. Kinetics of Escherichia coli B D-lactate dehydrogenase and evidence for pyruvate-controlled change in conformation. J Biol Chem. 1968 May 25;243(10):2587–2596. [PubMed] [Google Scholar]
  19. 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]
  20. Young M., Minton N. P., Staudenbauer W. L. Recent advances in the genetics of the clostridia. FEMS Microbiol Rev. 1989 Dec;5(4):301–325. doi: 10.1111/j.1574-6968.1989.tb03402.x. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES