Summary
Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNA originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.
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Abbreviations
- kop:
-
kilobase pairs
- OcTase:
-
ornithine carbamoyl transferase
- CPSase:
-
carbamoyl phosphate synthetase
References
Baumberg S (1976) Genetic control of arginine metabolism in prokaryotes. In: Macdonald KD (ed) Second international symposiun 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
Bolivar F (1978) Construction and characterisation of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant DNA molecules. Gene 4:121–136
Bolivar F, Backman K (1979) Plasmids of Escherichia coli as 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
Bretscher A, Baumberg S (1976) Divergent transcription of the arg ECBH cluster of Escherichia coli K12. Mutations which alter the control of enzyme synthesis. J Mol Biol 102:205–220
Chang AC, Cohen SN (1978) Construction and characterisation of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Chi N, Ehrlich SD, Lederberg J (1978) Functional expression of two Bacillus subtilis chromosomal genes in Escherichia coli. J Bacteriol 133:816–821
Flint H, Kemp B (1981) General control of arginine biosynthetic enzymes in Neurospora crassa. J Gen Microbiol 124:129–140
Gentz R, Langner A, Chang AC, Cohen SN, Bujard H (1981) Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal. Proc Natl Acad Sci USA 78:4936–4940
Hagan CE, Warren GJ (1983) Viability of palindromic DNA restored by deletions occurring at low frequency in plasmids of Escherichia coli. Gene 24:317–326
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
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
Ish-Horowicz D, Burke J (1981) Rapid and efficient cosmid cloning. Nucl Acid Res 9:2989–2998
Jones IM, Primrose SB, Ehrlich SD (1982) Recombination between short direct repeats in a recA host. Mol Gen Genet 188:486–489
Kelly MS (1967) Physical and mapping properties of distant linkage between genetic markers in transformation of Bacillus subtilis. Mol Gen Genet 99:333–349
Leemans J, Langenakens J, De Greve H, Van Montagu M, Schell J (1982) Broad lost range vectors derived from the W-plasmid Sa. Gene 19:361–364
Losick R, Pero J (1981) Cascades of sigma factors. Cell 25:582–584
Mandel M, Higa A (1970) A calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Mann NH, Mountain A, Munton RN, Smith MC, Baumberg S (1984) Cloning and transcription analysis of a Bacillus subtilis arg gene. Mol Gen Genet 197:75–81
Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218
McDonnell MW, Simon MN, Studier FW (1977) Analyses of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol 110:119–146
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
Nagahari K, Sakaguchi (1978) Cloning of Bacillus subtilis Leucine A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli. Mol Gen Genet 158:263–270
Neugebauer K, Sprengel R, Schaller H (1981) Penicillinase from Bacillus licheniformis: Nucleotide sequence of the gene and implications for biosynthesis of a secretory protein in a Grampositive bacterium. Nucl Acids Res 9:2577–2588
Palva L, pettersson RF, Kalkkinen N, Lehtovaara, Sarvas M, Soderlund H, Takkinen K, Kaariainen L (1981) Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the α-amylase gene from Bacillus amyloliquefaciens. Gene 15:43–51
Primrose SB, Ehrlich SD, (1981) Instability associated with deletion formation in a hybrid plasmid. Plasmid 6:193–201
Rapoport G, Klier A, Billault A, Fargette F, Dedonder R (1979) Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Mol Gen Genet 176:239–245
Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251
Seiki M, Ogasawara N, Yoshikawa H (1981a) Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. I. Isolation and characterisation of plasmids containing the origin. Mol Gen Genet 183:220–226
Seiki M, Ogasawara N, Yoshikawa H (1981b) Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. II. Identification of the essential regions for inhibitory functions shown by the DNA sequence containing the replication origin. Mol Gen Genet 183:227–233
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Stoker NG, Broome-Smith JK, Edelman A, Spratt BG (1983) Organisation and subcloning of the dacA-rodA-pbpA cluster of cell shape genes in Escherichia coli. J Bacteriol 155:847–853
Yang M, Galizzi A, Henner D (1983) Nucleotide sequence of the amylase gene from Bacillus subtilis. Nucl Acid Res 11:237–249
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Communicated by P.T. Emmerson
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Mountain, A., Mann, N.H., Munton, R.N. et al. Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis. Mol Gen Genet 197, 82–89 (1984). https://doi.org/10.1007/BF00327926
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DOI: https://doi.org/10.1007/BF00327926