Summary
Following shotgun cloning of EcoRI fragments of Bacillus subtilis DNA in pBR322, a hybrid plasmid pUL710 was isolated which complements argC but no other auxotrophs of E. coli K12. Restriction mapping, Southern blotting and other evidence suggest that pUL710 carries an insert of 1.6 kbp, and derives, by deletion of both vector and insert sequences, from a larger but unstable initial hybrid which carried a 12 kbp EcoRI fragment from the B. subtilis chromosome. RecE-dependent integration of pUL710 into the B. subtilis chromosome demonstrated homology between the insert DNA and the argO locus of B. subtilis. pUL710 was found to confer appreciable tetracycline resistance even though the deletion presumed to stabilise the hybrid had inactivated the tet promoter. The results of analysis by Tn5 mutagenesis, transcriptional fusions and run-off in vitro transcription suggest that both the cloned argC gene and the tetracycline gene in pUL710 are expressed from a B. subtilis promoter located very close to the EcoRI cloning site.
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Abbreviations
- AcGSA-DH:
-
N-acetylglutamic γ-semialdehyde dehydrogenase
References
Baumberg S (1976) Genetic control of arginine metabolism in prokaryotes. In: Macdonald KD (ed) Second international symposium on the genetics of industrial microorganisms. Academic Press, London, New York, pp 369–389
Baumberg S, Harwood CR (1979) Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis. J Bacteriol 119:189–196
Baumberg S, Mountain A (1984) B. subtilis 168 mutants resistant to arginine hydroxamate in the presence of ornithine or citrulline. J Gen Microbiol 130:1247–1252
Beeftinck F, Cunin R, Glansdorff N (1974) Arginine gene duplications in recombination proficient and deficient strains of Escherichia coli K12. Mol Gen Genet 132:241–253
Bolivar F, Backman K (1979) Plasmids of Escherichia coli cloning vectors. Methods Enzymol 68:245–267
Bolivar F, Rodriguez R, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterisation of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
Chang AC, Cohen SN (1978) Construction and characterisation of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid. J Bacteriol 134:1141–1150
Dooley DC, Hadden CT, Nester EW (1971) Macromolecular synthesis in B. subtilis in the competent state. J Bacteriol 108:668–679
Dubnau D, Cirigliano C (1974) Genetic characterisation of recombination-deficient mutants of Bacillus subtilis. J Bacteriol 117:488–493
Elseviers D, Cunin R, Glansdorff N, Baumberg S, Ashcroft E (1972) Control regions within the argECBH gene cluster of Escherichia coli. Mol Gen Genet 117:349–366
Ephrati-Elizur E (1968) Spontaneous transformation in Bacillus subtilis. Genet Res 11:83–96
Harwood CR, Baumberg S (1977) Arginine hydroxamate resistant mutants of Bacillus subtilis with altered control of arginine metabolism. J Gen Microbiol 100:177–188
Henner DJ, Hoch JA (1980) The Bacillus subtilis chromosome. Microbiol Rev 44:57–82
Hoopes BC, McClure WR (1981) Studies on the selectivity of DNA precipitation by spermine. Nucl Acid Res 9:5493–5504
Kehoe M, Sellwood R, Shipley P, Dougan G (1981) Genetic analysis of K88-mediated adhesion of enterotoxigenic Escherichia coli. Nature (Lond) 291:122–126
Kelly MS (1967) Physical and mapping properties of distant linkage between genetic markers in transformation of Bacillus subtilis. Mol Gen Genet 99:333–349
Kupper H, Contreras R, Landy R, Khorana HG (1975) Promoter-dependent transcription of tRNATyr genes using DNA fragments produced by restriction enzymes. Proc Natl Acad Sci USA 72:4754–4758
Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218
McMaster GK, Carmichael GG (1977) Analysis of single and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74:4835–4838
Mountain A, Baumberg S (1980) Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168. Mol Gen Genet 178:691–701
Mountain A, Mann NH, Munton RN, Baumberg S (1984) Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutations in eigh Escherichia coli genes of arginine biosynthesis. Mol Gen Genet 197:82–89
Rothestein SJ, Jorgensen RA, Postle K, Reznikoff WS (1980) The inverted repeats of Tn5 are functionally different. Cell 19:795–805
Sutcliffe JG (1979) The complete nucleotide sequence of Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–80
West RW, Rodriguez RL (1982) Constriction and characterisation of E. coli promoter-probe plasmid vectors. III. pBR322 derivatives with deletions in the tetracycline resistance promoter region. Gene 20:291–304
Young FE, Smith C, Reilly BE (1969) Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis. J Bacteriol 98:1087–1097
Young M (1983) The mechanism of insertion of a segment of heterologous DNA into the chromosome of Bacillus subtilis. J Gen Microbiol 129:1497–1512
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Communicated by P.T. Emmerson
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Mann, N.H., Mountain, A., Munton, R.N. et al. Transcription analysis of a Bacillus subtilis arg gene following cloning in Escherichia coli in an initially unstable hybrid plasmid. Mol Gen Genet 197, 75–81 (1984). https://doi.org/10.1007/BF00327925
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DOI: https://doi.org/10.1007/BF00327925