Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Jun;4(6):1599–1603. doi: 10.1002/j.1460-2075.1985.tb03823.x

Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

E Schwarz, D Oesterhelt
PMCID: PMC554388  PMID: 3896775

Abstract

Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.

Full text

PDF
1599

Images in this article

1601Fig. 3.
on p.1601
1602Fig. 4.
on p.1602

Selected References

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

  1. Buckel W., Semmler R. Purification, characterisation and reconstitution of glutaconyl-CoA decarboxylase, a biotin-dependent sodium pump from anaerobic bacteria. Eur J Biochem. 1983 Nov 2;136(2):427–434. doi: 10.1111/j.1432-1033.1983.tb07760.x. [DOI] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collins J. Escherichia coli plasmids packageable in vitro in lambda bacteriophage particles. Methods Enzymol. 1979;68:309–326. doi: 10.1016/0076-6879(79)68022-9. [DOI] [PubMed] [Google Scholar]
  5. Dimroth P. A new sodium-transport system energized by the decarboxylation of oxaloacetate. FEBS Lett. 1980 Dec 29;122(2):234–236. doi: 10.1016/0014-5793(80)80446-7. [DOI] [PubMed] [Google Scholar]
  6. Dimroth P. Characterization of a membrane-bound biotin-containing enzyme: oxaloacetate decarboxylase from Klebsiella aerogenes. Eur J Biochem. 1981 Apr;115(2):353–358. doi: 10.1111/j.1432-1033.1981.tb05245.x. [DOI] [PubMed] [Google Scholar]
  7. Dimroth P. The generation of an electrochemical gradient of sodium ions upon decarboxylation of oxaloacetate by the membrane-bound and Na+-activated oxaloacetate decarboxylase from Klebsiella aerogenes. Eur J Biochem. 1982 Jan;121(2):443–449. doi: 10.1111/j.1432-1033.1982.tb05807.x. [DOI] [PubMed] [Google Scholar]
  8. Dimroth P., Thomer A. Subunit composition of oxaloacetate decarboxylase and characterization of the alpha chain as carboxyltransferase. Eur J Biochem. 1983 Dec 1;137(1-2):107–112. doi: 10.1111/j.1432-1033.1983.tb07802.x. [DOI] [PubMed] [Google Scholar]
  9. Hall B. G. Chromosomal mutation for citrate utilization by Escherichia coli K-12. J Bacteriol. 1982 Jul;151(1):269–273. doi: 10.1128/jb.151.1.269-273.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hertzberg E. L., Hinkle P. C. Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli. Biochem Biophys Res Commun. 1974 May 7;58(1):178–184. doi: 10.1016/0006-291x(74)90908-5. [DOI] [PubMed] [Google Scholar]
  11. Hilpert W., Dimroth P. Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens. Eur J Biochem. 1983 May 16;132(3):579–587. doi: 10.1111/j.1432-1033.1983.tb07403.x. [DOI] [PubMed] [Google Scholar]
  12. Hilpert W., Schink B., Dimroth P. Life by a new decarboxylation-dependent energy conservation mechanism with Na as coupling ion. EMBO J. 1984 Aug;3(8):1665–1670. doi: 10.1002/j.1460-2075.1984.tb02030.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Reynolds C. H., Silver S. Citrate utilization by Escherichia coli: plasmid- and chromosome-encoded systems. J Bacteriol. 1983 Dec;156(3):1019–1024. doi: 10.1128/jb.156.3.1019-1024.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shinagawa M., Makino S., Hirato T., Ishiguro N., Sato G. Comparison of DNA sequences required for the function of citrate utilization among different citrate utilization plasmids. J Bacteriol. 1982 Aug;151(2):1046–1050. doi: 10.1128/jb.151.2.1046-1050.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Vogelsang H., Oertel W., Oesterhelt D. Isolation of the bacterioopsin gene by colony hybridization. Methods Enzymol. 1983;97:226–241. doi: 10.1016/0076-6879(83)97135-5. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES