CLONING OF BACILLUS SUBTILIS DNA: EXPRESSION IN B. SUBTILIS AND ESCHERICHIA COLI.

Abstract

Bacillus subtilis DNA was cloned by ligating restriction endonuclease-generated fragments to plasmid vectors. The plasmid pUB110 was the vehicle in the construction of eight recombinant plasmids, pNM1 through pNM8. Each bears one or more EcoRI fragment(s) of B. subtilis chromosomal DNA. Recovery of the plasmids from host cells demonstrated that recombinant plasmids that bear some homology to the B, subtilis chromosome may be maintained outside of the chromosome in recombination-proficient hosts. The mean size of cloned fragments was 0.78 megadaltons (Mdal). The recombinant plasmid pNM1 interferes with the mechanism that blocks chromosomal recombination in B. subtilis cells that carry the recE4 mutation. Low-level chromosomal recombination at several loci was demonstrated when chromosomal DNA was accompanied by pNM1 in the transformation of recE4 recipient cells. The recombinant plasmid does not appear to code for recE gene products nor does it produce novel proteins when assayed in minicells of B. subtilis. An alternative approach to cloning B. subtilis DNA was successfully accomplished with the vector plasmid pHV33. The vector functions in both B. subtilis and E. coli hosts. B. subtilis chromosomal DNA was digested with Bg1II, then ligated to the unique BamHI site of pHV33. Ligation products were introduced into E. coli by transformation. Plasmid DNAs were isolated from transformants, pooled into several lots, then used to transform auxotrophic B. subtilis recipient cells. The procedure resulted in the construction of two new recombinant plasmids, pNM1055 and pNM1326. B. subtilis cells with the aroD120 mutation restored their ability to synthesize aromatic amino acids when pNM1055 was introduced. The same effect was observed in E. coli recipient cells that had the equivalent mutation. E. coli cells that carried pNM1326 produced granular colonies characteristic of the extraordinary filamentous growth exhibited by individual cells. The pNM1326 plasmid coded for a 16,000 dalton polypeptide produced in abundant quantities in E. coli hosts. A deletion derivative of pNM1326 did not produce the polypeptide, nor was filamentous growth of host cells exhibited. A plasmid-borne fragment of B. subtilis DNA affects cells growth and division of E. coli hosts.