DNA cloning in Bacillus subtilis ( plasmids / recombinant molecules / heterospecific gene expression ) STANISLAV

A plasmid pC194, encoding resistance to chloramphenicol, can serve as a cloning vector in Bacillus subtilis 168 for other HindIII-cleaved DNA segments. Replicons constructed by linking pC194 to several Escherichia coli plasmids can be used to introduce and compare the expression of the same genes in these two bacterial hosts. Since the pioneering experiments of Cohen and his colleagues (1), the technology of DNA cloning in Escherichia coli has become a major tool of molecular genetics. The main extensions beyond E. coli experiments are with plasmid RP4 and its wide range of Gram-negative hosts (2). Bacillus subtilis is a Gram-positive, sporulating, aerobic bacterium phylogenetically very distant from E. coli. A B. subtilis cloning system (3) parallel to that developed for E. coli permits studies of the interaction of the same genes with two, possibly very different, cellular environments, which would offer greater insight into the diverse processes that convert genetic information to the corresponding phenotype. The development of the B. subtilis cloning system has been mainly hindered by the absence of suitable vector replicons. Several plasmids can be introduced and maintained in the highly transformable B. subtilis strain 168 (4, 5); however, they lacked genetic markers for selection and DNA cloning. More recently, plasmids originally isolated from Staphylococcus aureus that code for resistance to tetracycline or chloramphenicol have been transfected into B. subtilis (6). These can be tested for their ability to accept inserted DNA segments, while still maintaining the capacity to replicate and express their genetic information in this host. The present experiments show that DNA segments can be inserted into the chloramphenicol resistance plasmid pC194, which then constitutes a DNA cloning vector for B. subtilis 168. MATERIALS AND METHODS Bacterial Strains. E. coli strains used were W5443 thr-1 leu-6 thi-l supE44 lacYl rmstrR (tonB tryp delta). The following plasmids were carried in the C600 strain: pSC105 (1), pBR322 (7), pBR313 (8), pMB9 (8), and pWL7 (from W. Goebel). B. subtilis strains included SB202 trypC2 his-2 tyr-1 aroB and SB634 thy aroB tyr-l, which were deposited in the collection of this laboratory. The plasmids pT127 and pC194 (6) were carried by SB634. DNAs and Enzymes. Plasmid DNAs were prepared by the cleared lysis procedure (9). The HindIII, BamHI, and Pst restriction endonucleases were commercial preparations (Biolabs, Beverly, MA). Hae III was a gift from V. Sgaramella. The EcoRI endonuclease and the T4 DNA ligase were isolated and used as described (10, 11). Transformation Procedure. Competence-induction and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advrtisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1433 transformation of E. coli and B. subtilis strains were done by published procedures (12, 13). Potential biohazards associated with the experiments described in this publication have been reviewed by the French National Control Committee. The appropriate experiments were performed at P2 containment level. Electrophoresis. Endonuclease-digested DNA was separated on horizontal agarose gels essentially as described (14).