Biopolym. Cell. 2000; 16(5):403-408.
http://dx.doi.org/10.7124/bc.000582
Cell Biology
Replacement of groEL gene of Escherichia coli by its homologue from Sinorhizobium meliloti
1Yerko V. N., 2Lund P. A.
  1. Institute of Plant Physiology and Genetics, NAS of Ukraine
    31/17, Vasylkivska, Kyiv, Ukraine, 03022
  2. School of Biosciences, The University of Birmingham
    Birmingham, B15 2TT, UK

Abstract

We show that groEL gene from S. meliloti coding for chaperonin's subunits with molecular weight 60 kDa complements deletion of groEL gene of E. coli accomplished by replacement of groEL by nptll gene using PI phage transduction. For expression of groEL gene from S. meliloti in E. coli cells we used plasmid with E. coli arabinose promoter allowing tight regulation of chaperonin biosynthesis by varying concentration of arabinose and glucose in growth medium. The method of replacement of chromosomal groEL gene by heterologous groEL allows investigation of chaperonin's behavior in a cell that does not carry additionally temperature sensitive chaperonins and thus allows to avoid undesirable interaction of chaperonins of differen origin and inclusion of heterologous subunits into the same chaperonin.
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References

[1] Hartl FU, Martin J. Molecular chaperones in cellular protein folding. Curr Opin Struct Biol. 1995;5(1):92-102.
[2] McLennan NF, McAteer S, Masters M. The tail of a chaperonin: the C-terminal region of Escherichia coli GroEL protein. Mol Microbiol. 1994;14(2):309-21.
[3] H?bner P, Dame G, Sandmeier U, Vandekerckhove J, Beyer P, Tadros MH. Molecular analysis of the Rhodobacter capsulatus chaperonin (groESL) operon: purification and characterization of Cpn60. Arch Microbiol. 1996;166(3):193-203.
[4] Rusanganwa E, Gupta RS. Cloning and characterization of multiple groEL chaperonin-encoding genes in Rhizobium meliloti. Gene. 1993;126(1):67-75.
[5] Ogawa J, Long SR. The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD. Genes Dev. 1995;9(6):714-29.
[6] Wallington EJ, Lund PA. Rhizobium leguminosarum contains multiple chaperonin (cpn60) genes. Microbiology. 1994;140 ( Pt 1):113-22.
[7] Yerko VN, Lund PA. Gene cpn60-1 coding one of homologous chaperonins in Rhizobiuin leguminosarum is essential for cell life. Biopolym Cell. 1999; 15(6):516-21.
[8] Gibson T. PhD thesis. Cambridge: Univ. press, 1997.
[9] Ivic A, Olden D, Wallington EJ, Lund PA. Deletion of Escherichia coli groEL is complemented by a Rhizobium leguminosarum groEL homologue at 37 degrees C but not at 43 degrees C. Gene. 1997;194(1):1-8.
[10] Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995;177(14):4121-30.
[11] Miller JH. Short course in bacterial genetics. New York: Cold Spring Harbor Lab. press, 1992.
[12] Sambrook J, Maniatis T, Fritsch EF. Molecular Cloning. A Laboratory Manual. New York: Cold Spring Harbor Lab. press, 1989.
[13] Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7(6):1513-23.