Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 May;173(10):3084–3095. doi: 10.1128/jb.173.10.3084-3095.1991

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

K R Hancock 1, E Rockman 1, C A Young 1, L Pearce 1, I S Maddox 1, D B Scott 1
PMCID: PMC207901  PMID: 1850729

Abstract

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.

Full text

PDF
3084

Images in this article

3088Image
on p.3088

Selected References

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

  1. Berg D. E., Davies J., Allet B., Rochaix J. D. Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3628–3632. doi: 10.1073/pnas.72.9.3628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergquist P. L., Adelberg E. A. Abnormal excision and transfer of chromosomal segments by a strain of Escherichia coli K-12. J Bacteriol. 1972 Jul;111(1):119–128. doi: 10.1128/jb.111.1.119-128.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Buvinger W. E., Riley M. Nucleotide sequence of Klebsiella pneumoniae lac genes. J Bacteriol. 1985 Sep;163(3):850–857. doi: 10.1128/jb.163.3.850-857.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Crow V. L., Thomas T. D. Properties of a Streptococcus lactis strain that ferments lactose slowly. J Bacteriol. 1984 Jan;157(1):28–34. doi: 10.1128/jb.157.1.28-34.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dills S. S., Apperson A., Schmidt M. R., Saier M. H., Jr Carbohydrate transport in bacteria. Microbiol Rev. 1980 Sep;44(3):385–418. doi: 10.1128/mr.44.3.385-418.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dixon R., Cannon F., Kondorosi A. Construction of a P plasmid carrying nitrogen fixation genes from Klebsiella pneumoniae. Nature. 1976 Mar 18;260(5548):268–271. doi: 10.1038/260268a0. [DOI] [PubMed] [Google Scholar]
  13. Egelhoff T. T., Long S. R. Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti. J Bacteriol. 1985 Nov;164(2):591–599. doi: 10.1128/jb.164.2.591-599.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  15. Garnier T., Cole S. T. Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens. Plasmid. 1988 Mar;19(2):134–150. doi: 10.1016/0147-619x(88)90052-2. [DOI] [PubMed] [Google Scholar]
  16. Graves M. C., Rabinowitz J. C. In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms. J Biol Chem. 1986 Aug 25;261(24):11409–11415. [PubMed] [Google Scholar]
  17. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  19. Herman R. E., McKay L. L. Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli. Appl Environ Microbiol. 1986 Jul;52(1):45–50. doi: 10.1128/aem.52.1.45-50.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herrchen M., Legler G. Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli. Eur J Biochem. 1984 Feb 1;138(3):527–531. doi: 10.1111/j.1432-1033.1984.tb07947.x. [DOI] [PubMed] [Google Scholar]
  21. Hirata H., Fukazawa T., Negoro S., Okada H. Structure of a beta-galactosidase gene of Bacillus stearothermophilus. J Bacteriol. 1986 Jun;166(3):722–727. doi: 10.1128/jb.166.3.722-727.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hirata H., Negoro S., Okada H. Molecular basis of isozyme formation of beta-galactosidases in Bacillus stearothermophilus: isolation of two beta-galactosidase genes, bgaA and bgaB. J Bacteriol. 1984 Oct;160(1):9–14. doi: 10.1128/jb.160.1.9-14.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  24. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Janssen P. J., Jones W. A., Jones D. T., Woods D. R. Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum. J Bacteriol. 1988 Jan;170(1):400–408. doi: 10.1128/jb.170.1.400-408.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jefferson R. A., Burgess S. M., Hirsh D. beta-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8447–8451. doi: 10.1073/pnas.83.22.8447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Kalnins A., Otto K., Rüther U., Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2(4):593–597. doi: 10.1002/j.1460-2075.1983.tb01468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kleckner N. Regulating tn10 and is10 transposition. Genetics. 1990 Mar;124(3):449–454. doi: 10.1093/genetics/124.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. McLaughlin J. R., Murray C. L., Rabinowitz J. C. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283–11291. [PubMed] [Google Scholar]
  32. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  33. Needleman S. B., Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443–453. doi: 10.1016/0022-2836(70)90057-4. [DOI] [PubMed] [Google Scholar]
  34. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schmidt B. F., Adams R. M., Requadt C., Power S., Mainzer S. E. Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli. J Bacteriol. 1989 Feb;171(2):625–635. doi: 10.1128/jb.171.2.625-635.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Stokes H. W., Betts P. W., Hall B. G. Sequence of the ebgA gene of Escherichia coli: comparison with the lacZ gene. Mol Biol Evol. 1985 Nov;2(6):469–477. doi: 10.1093/oxfordjournals.molbev.a040372. [DOI] [PubMed] [Google Scholar]
  41. Stormo G. D., Schneider T. D., Gold L. M. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. doi: 10.1093/nar/10.9.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thuring R. W., Sanders J. P., Borst P. A freeze-squeeze method for recovering long DNA from agarose gels. Anal Biochem. 1975 May 26;66(1):213–220. doi: 10.1016/0003-2697(75)90739-3. [DOI] [PubMed] [Google Scholar]
  43. Usdin K. P., Zappe H., Jones D. T., Woods D. R. Cloning, Expression, and Purification of Glutamine Synthetase from Clostridium acetobutylicum. Appl Environ Microbiol. 1986 Sep;52(3):413–419. doi: 10.1128/aem.52.3.413-419.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Whitfeld P. L., Seeburg P. H., Shine J. The human pro-opiomelanocortin gene: organization, sequence, and interspersion with repetitive DNA. DNA. 1982;1(2):133–143. doi: 10.1089/dna.1.1982.1.133. [DOI] [PubMed] [Google Scholar]
  45. Wilbur W. J., Lipman D. J. Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci U S A. 1983 Feb;80(3):726–730. doi: 10.1073/pnas.80.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Youngleson J. S., Jones W. A., Jones D. T., Woods D. R. Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum. Gene. 1989 May 30;78(2):355–364. doi: 10.1016/0378-1119(89)90238-2. [DOI] [PubMed] [Google Scholar]
  48. Youngleson Jonathan S., Santangelo Joseph D., Jones David T., Woods David R. Cloning and Expression of a Clostridium acetobutylicum Alcohol Dehydrogenase Gene in Escherichia coli. Appl Environ Microbiol. 1988 Mar;54(3):676–682. doi: 10.1128/aem.54.3.676-682.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zappe H., Jones D. T., Woods D. R. Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli. J Gen Microbiol. 1986 May;132(5):1367–1372. doi: 10.1099/00221287-132-5-1367. [DOI] [PubMed] [Google Scholar]

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

RESOURCES