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. 1987 Apr;55(4):848–855. doi: 10.1128/iai.55.4.848-855.1987

Cloning and functional expression of the Coxiella burnetii citrate synthase gene in Escherichia coli.

R A Heinzen, L P Mallavia
PMCID: PMC260428  PMID: 3104207

Abstract

The citrate synthase gene from the obligate intracellular rickettsial parasite Coxiella burnetii was cloned and expressed in Escherichia coli. Transduction into E. coli with a C. burnetii gene library constructed in the cosmid vector pHK17 resulted in the functional complementation of the gltA mutation of E. coli MOB154. A GltA+ clone carrying 16.4 kilobase pairs of C. burnetii DNA and designated pJCC959 was isolated and characterized. Southern hybridization analysis confirmed that the pJCC959 cloned insert consists of C. burnetii DNA and that homology exists with the Rickettsia prowazekii citrate synthase gene. Subcloning analysis with the multicopy expression vector pUC8 revealed that citrate synthase expression was under control of a C. burnetti promoter. In vitro transcription-translation of subclones pLPM20 and pLPM30 established a molecular weight of ca. 46,000 for the monomer form of the cloned enzyme. Transposon Tn5 mutagenesis of pLPM30 defined the coding region to approximately 1.2 kilobase pairs of C. burnetii DNA. Maxicell analysis of selected pLPM30::Tn5 insertion derivatives identified the direction of transcription and the relative translational start and stop sites and substantiated the molecular weight value calculated from the in vitro analysis. Inhibition studies showed that citrate synthase activity in crude cell extracts obtained from strain MOB154 transformed with the cloned C. burnetii gene was markedly inhibited by 4 mM ATP, while 4 mM alpha-ketoglutarate had virtually no effect. These data indicate that the C. burnetii enzyme displays regulatory behavior characteristic of the small gram-positive bacterial and eucaryotic enzyme.

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Selected References

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  1. Auerswald E. A., Ludwig G., Schaller H. Structural analysis of Tn5. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):107–113. doi: 10.1101/sqb.1981.045.01.019. [DOI] [PubMed] [Google Scholar]
  2. Baca O. G., Paretsky D. Q fever and Coxiella burnetii: a model for host-parasite interactions. Microbiol Rev. 1983 Jun;47(2):127–149. doi: 10.1128/mr.47.2.127-149.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhayana V., Duckworth H. W. Amino acid sequence of Escherichia coli citrate synthase. Biochemistry. 1984 Jun 19;23(13):2900–2905. doi: 10.1021/bi00308a008. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bittner M., Kupferer P., Morris C. F. Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal Biochem. 1980 Mar 1;102(2):459–471. doi: 10.1016/0003-2697(80)90182-7. [DOI] [PubMed] [Google Scholar]
  6. Bloxham D. P., Parmelee D. C., Kumar S., Walsh K. A., Titani K. Complete amino acid sequence of porcine heart citrate synthase. Biochemistry. 1982 Apr 27;21(9):2028–2036. doi: 10.1021/bi00538a009. [DOI] [PubMed] [Google Scholar]
  7. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  8. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  9. Burton P. R., Stueckemann J., Welsh R. M., Paretsky D. Some ultrastructural effects of persistent infections by the rickettsia Coxiella burnetii in mouse L cells and green monkey kidney (Vero) cells. Infect Immun. 1978 Aug;21(2):556–566. doi: 10.1128/iai.21.2.556-566.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DAVIS B. D., GILVARG C. The role of the tricarboxylic acid cycle in acetate oxidation in Escherichia coli. J Biol Chem. 1956 Sep;222(1):307–319. [PubMed] [Google Scholar]
  11. Giphart-Gassler M., Plasterk R. H., van de Putte P. G inversion in bacteriophage Mu: a novel way of gene splicing. Nature. 1982 May 27;297(5864):339–342. doi: 10.1038/297339a0. [DOI] [PubMed] [Google Scholar]
  12. Hackstadt T., Williams J. C. Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc Natl Acad Sci U S A. 1981 May;78(5):3240–3244. doi: 10.1073/pnas.78.5.3240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hackstadt T., Williams J. C. Stability of the adenosine 5'-triphosphate pool in Coxiella burnetii: influence of pH and substrate. J Bacteriol. 1981 Nov;148(2):419–425. doi: 10.1128/jb.148.2.419-425.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hackstadt T., Williams J. C. pH dependence of the Coxiella burnetii glutamate transport system. J Bacteriol. 1983 May;154(2):598–603. doi: 10.1128/jb.154.2.598-603.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hendrix L., Mallavia L. P. Active transport of proline by Coxiella burnetii. J Gen Microbiol. 1984 Nov;130(11):2857–2863. doi: 10.1099/00221287-130-11-2857. [DOI] [PubMed] [Google Scholar]
  16. Jangaard N. O., Unkeless J., Atkinson D. E. The inhibition of citrate synthase by adenosine triphosphate. Biochim Biophys Acta. 1968 Jan 8;151(1):225–235. doi: 10.1016/0005-2744(68)90177-0. [DOI] [PubMed] [Google Scholar]
  17. Kleckner N., Chan R. K., Tye B. K., Botstein D. Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition. J Mol Biol. 1975 Oct 5;97(4):561–575. doi: 10.1016/s0022-2836(75)80059-3. [DOI] [PubMed] [Google Scholar]
  18. Klee H. J., Gordon M. P., Nester E. W. Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity. J Bacteriol. 1982 Apr;150(1):327–331. doi: 10.1128/jb.150.1.327-331.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. McDonald T. L., Mallavia L. Biochemistry of Coxiella burnetii: Embden-Meyerhof pathway. J Bacteriol. 1971 Sep;107(3):864–869. doi: 10.1128/jb.107.3.864-869.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mergeay M., Gigot D., Beckmann J., Glansdorff N., Piérard A. Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12. Mol Gen Genet. 1974;133(4):299–316. doi: 10.1007/BF00332706. [DOI] [PubMed] [Google Scholar]
  22. Phibbs P. V., Jr, Winkler H. H. Regulatory properties of citrate synthase from Rickettsia prowazekii. J Bacteriol. 1982 Feb;149(2):718–725. doi: 10.1128/jb.149.2.718-725.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Remington S., Wiegand G., Huber R. Crystallographic refinement and atomic models of two different forms of citrate synthase at 2.7 and 1.7 A resolution. J Mol Biol. 1982 Jun 15;158(1):111–152. doi: 10.1016/0022-2836(82)90452-1. [DOI] [PubMed] [Google Scholar]
  24. STOENNER H. G., LACKMAN D. B. The biologic properties of Coxiella burnetii isolated from rodents collected in Utah. Am J Hyg. 1960 Jan;71:45–51. doi: 10.1093/oxfordjournals.aje.a120088. [DOI] [PubMed] [Google Scholar]
  25. Samuel J. E., Frazier M. E., Kahn M. L., Thomashow L. S., Mallavia L. P. Isolation and characterization of a plasmid from phase I Coxiella burnetii. Infect Immun. 1983 Aug;41(2):488–493. doi: 10.1128/iai.41.2.488-493.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Samuel J. E., Frazier M. E., Mallavia L. P. Correlation of plasmid type and disease caused by Coxiella burnetii. Infect Immun. 1985 Sep;49(3):775–779. doi: 10.1128/iai.49.3.775-779.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sancar A., Wharton R. P., Seltzer S., Kacinski B. M., Clarke N. D., Rupp W. D. Identification of the uvrA gene product. J Mol Biol. 1981 May 5;148(1):45–62. doi: 10.1016/0022-2836(81)90234-5. [DOI] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  29. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  30. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  31. Weitzman P. D., Danson M. J. Citrate synthase. Curr Top Cell Regul. 1976;10:161–204. doi: 10.1016/b978-0-12-152810-2.50011-5. [DOI] [PubMed] [Google Scholar]
  32. Weitzman P. D., Jones D. Regulation of citrate synthase and microbial taxonomy. Nature. 1968 Jul 20;219(5151):270–272. doi: 10.1038/219270a0. [DOI] [PubMed] [Google Scholar]
  33. Winkler H. H. Rickettsial permeability. An ADP-ATP transport system. J Biol Chem. 1976 Jan 25;251(2):389–396. [PubMed] [Google Scholar]
  34. Wood D. O., Atkinson W. H., Sikorski R. S., Winkler H. H. Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coli. J Bacteriol. 1983 Jul;155(1):412–416. doi: 10.1128/jb.155.1.412-416.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wright J. A., Maeba P., Sanwal B. D. Allosteric regulation of the activity of citrate snythetase of Escherichia coli by alpha-ketoglutarate. Biochem Biophys Res Commun. 1967 Oct 11;29(1):34–38. doi: 10.1016/0006-291x(67)90536-0. [DOI] [PubMed] [Google Scholar]
  36. de Bruijn F. J., Lupski J. R. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids--a review. Gene. 1984 Feb;27(2):131–149. doi: 10.1016/0378-1119(84)90135-5. [DOI] [PubMed] [Google Scholar]

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