Cloning restriction fragments that promote expression of a gene in Bacillus subtilis

Plasmid pPL603 (3.1 megadaltons) specifies neomycin resistance in Bacillus subtilis and contains a structural gene for chloramphenicol acetyltransferase. Cells harboring the plasmid cannot grow on solid media containing 10 microgram of chloramphenicol per ml. Cloning EcoRI (or EcoRI)-generated fragments of deoxyribonucleic acid from several sources into the single EcoRI site in plasmid pPL603, with subsequent selection of transformants of media containing 10 micrograms of chloramphenicol per ml, permits the identification of restriction fragments that promote expression of the chloramphenicol acetyltransferase gene.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

Isolation of an autonomously replicating DNA fragment from the region of defective bacteriophage PBSX of Bacillus subtilis

We have isolated a 5.4-kilobase fragment of Bacillus subtilis DNA that confers the ability to replicate upon a nonreplicative plasmid. The B. subtilis 168 EcoRI fragment was ligated into the chimeric plasmid pCs540, which contains a chloramphenicol resistance determinant from the Staphylococcus aureus plasmid pC194 and an HpaII fragment from the Escherichia coli plasmid, pSC101. A recE B. subtilis derivative, strain BD224, is capable of maintaining this DNA as an autonomously replicating plasmid. In rec+ recipients, chloramphenicol-resistant transformants do not contain free plasmid. The plasmid is integrated as demonstrated by alterations in the pattern of chromosomal restriction enzyme fragments to which the plasmid hybridizes. The site of plasmid integration was mapped by PBS1-mediated transduction to the metC-PBSX region. A strain was a deletion in the region of defective bacteriophage PBSX differs in the hybridization profile obtained by probing EcoRI digests with this cloned fragment. This same deletion mutant, though proficient in normal recombinational pathways, permits autonomous replication of the plasmid apparently owing to the lack of an homologous chromosomal region with which to recombine. We believe that, like E. coli. B. subtilis contains at least one DNA fragment capable of autonomous replication when liberated from its normally integrated chromosomal site and that this cloned DNA fragment comes from the region of defective bacteriophage PBSX.     

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.     

Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.     

High copy number plasmid vectors for use in lactic streptococci

Abstract A 3.8 kb DNA fragment from plasmid pBD64 which encoded chloramphenicol and kanamycin resistance genes, but had no replication region, was used as a replicator probe to select for the replication region of the cryptic lactic streptococcal plasmid pSH71 using Bacillus subtilis as host. Three of the resultant recombinant plasmids, pCK1, pCK17 and pCK21 are described. They are vectors in Streptococcus lactis and can be used to clone Bgl II-compatible fragments into their kanamycin resistance gene. All the plasmids have single sites for restriction endonucleases Ava I, Bam HI, Eco RI, Pvu II and Xba I, while plasmids pCK17 and pCK21 have single sites for Cla I.     

Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.     

Expression of the Bacillus pumilus chloramphenicol acetyltransferase gene in Bacillus subtilis, achieved by the P-R-promotor of phage lambda

The possibility of expression of the Bacillus pumilus chloramphenicol acetyltransferase gene (cat) in Bacillus subtilis from the pR promoter of phage lambda has been investigated in this work. For this purpose, the plasmid pPL703 carrying the B. pumilus DNA segment with the cat gene lacking promoter has been combined with the plasmid pBM21 containing the pR promoter. The recombinant plasmid pEL1 is capable of providing the 60 mkg/ml chloramphenicol resistance in Bac. subtilis cells.     

Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid

A promoter-probe plasmid suitable for use in Xanthomonas campestris pathovar campestris (causal agent of crucifer black rot) was constructed by ligating a broad host range IncQ replicon into the promoter-probe plasmid pKK232-8, which contains a promoterless chloramphenicol acetyltransferase gene. Xanthomonas chromosomal DNA fragments were 'shotgun' cloned into a restriction site in front of this gene, and the resulting library was transferred en masse into Xanthomonas. Individual transconjugants possessing DNA insertions with promoter activity in plants were identified by virtue of their ability to infect chloramphenicol-treated turnip seedlings. Of 19 transconjugants identified in this way five were chloramphenicol resistant both in turnip seedlings and on agar plates. However the remaining 14 were only chloramphenicol resistant in planta, and thus apparently contained plant-inducible promoter fragments. Resistance to chloramphenicol was correlated with increased chloramphenicol acetyltransferase activity for the transconjugants assayed. The promoter fragments were used to isolate genomic clones from a library, and the role of the genes contained in these clones in pathogenicity is being investigated.