Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

New plasmid expression vectors for Bacillus subtilis

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.     

Construction of hybrid plasmid vectors for cloning inEscherichia coli,Bacillus subtilis,and the cyanobacteriumAnacystis nidulans

Two hybrid plasmids capable of acting as shuttle cloning vectors inAnacystis nidulans andBacillus subtilis were constructed by in vitro ligation. One construct, pMG202, consists of theB. subtilis vector pNN101 and the endogenous cyanobacterial plasmid pUH24. This 14.6 kb plasmid confers chloramphenicol resistance in both hosts and tetracycline resistance inB. subtilis. A second vector, pMG101, consists of pNN101 linked to theA. nidulans-Escherichia coli chimeric plasmid pCB4 and is 12.9 kb in size. The pCB4 portion of the vector enables pMG101 to replicate in the third host,E. coli, and confers ampicillin resistance in this bacterium as well as inA. nidulans. Both plasmids possess identical uniqueStu I sites which permit insertional inactivation of the chloramphenicol resistance gene; and, in addition, identical uniqueXho I sites are present on both vectors. Each vector also has a third unique site:Sma I on pMG101 andXba I on pMG202.     

Structurally stable Bacillus subtilis cloning vectors

Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does. In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.     

Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli

The cryptic Streptococcus cremoris Wg2 plasmid pWV01 (1.5 megadaltons) was genetically marked with the chloramphenicol resistance (Cmr) gene from pC194. The recombinant plasmid (pGK1, 2.4 megadaltons) replicated and expressed Cmr in Bacillus subtilis. From this plasmid an insertion-inactivation vector was constructed by inserting the erythromycin resistance (Emr) gene from pE194 cop-6. This plasmid (pGK12, 2.9 megadaltons) contained a unique BclI site in the Emr gene and unique ClaI and HpaII sites outside both resistance genes. It was stably maintained in B. subtilis at a copy number of approximately 5. pGK12 also transformed Escherichia coli competent cells to Cmr and Emr. The copy number in E. coli was about 60. Moreover, pGK12 transformed protoplasts of Streptococcus lactis. In this host both resistance genes are expressed. pGK12 is stably maintained in S. lactis at a copy number of 3.     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA

Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.     

Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli.

The cryptic Streptococcus cremoris Wg2 plasmid pWV01 (1.5 megadaltons) was genetically marked with the chloramphenicol resistance (Cmr) gene from pC194. The recombinant plasmid (pGK1, 2.4 megadaltons) replicated and expressed Cmr in Bacillus subtilis. From this plasmid an insertion-inactivation vector was constructed by inserting the erythromycin resistance (Emr) gene from pE194 cop-6. This plasmid (pGK12, 2.9 megadaltons) contained a unique BclI site in the Emr gene and unique ClaI and HpaII sites outside both resistance genes. It was stably maintained in B. subtilis at a copy number of approximately 5. pGK12 also transformed Escherichia coli competent cells to Cmr and Emr. The copy number in E. coli was about 60. Moreover, pGK12 transformed protoplasts of Streptococcus lactis. In this host both resistance genes are expressed. pGK12 is stably maintained in S. lactis at a copy number of 3.     

R391: a conjugative integrating mosaic comprised of phage,plasmid, and transposon elements

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.     

Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis.

Restriction endonuclease cleavage maps of seven chimeric plasmids that may be used for molecular cloning in Bacillus subtilis are presented. These plasmids all carry multiple antibiotic resistance markers and were constructed by in vitro molecular cloning techniques. Several of the antibiotic resistance markers were shown to undergo insertional inactivation at specific restriction endonuclease sites. Kanamycin inactivation occurred at the BglII site of pUB110 derivatives, erythromycin inactivation occurred at the HpaI and BclI sites of pE194 derivatives, and streptomycin inactivation occurred at the HindIII site of pSA0501 derivatives. A stable mini-derivative of pBD12 was isolated and characterized. By using these plasmids, we identified proteins involved in plasmid-coded kanamycin and erythromycin resistance. The properties and uses of these chimeric plasmids in the further development of recombinant deoxyribonucleic acid technology in B. subtilis are discussed.     

Characterization and cloning of gene 5 of Bacillus subtilis phage phi 29

Sequencing of the phi 29 DNA region [open reading frames (ORFs) 12, 11 and 10] between genes 6 and 4 of the mutant ts5(219) showed that a G in the wild-type phage had been changed to an A in the mutant at position 218 of ORF 10 indicating that this ORF corresponds to gene 5. ORF 10 was cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda and, after heat induction of the Escherichia coli cells carrying the recombinant plasmid pGM26, a 12-kDa protein was overproduced, accounting for about 5% of the de novo synthesized protein. Introduction of a nonsense mutation in ORF 10 indicated that the latter codes for the 12-kDa protein. The predicted secondary structure, the hydrophilicity values and the antigenic regions of protein p5 are discussed.     

Construction of new shuttle plasmid vectors for Escherichia coli-Bacteroides transgeneric cloning

Abstract An Escherichia coli-Bacteroides shuttle vehicle (pKBF367-1) was constructed by combining the pBR322 derivative pKC7 (5.9 kb) with [1] a 4.6 kb cryptic plasmid from Bacteroides fragilis ; and [2] the 4.2 kb Eco RI-B fragment of the B. fragilis plasmid pBFTM10. This latter component allowed selection of clindamycin-resistant transconjugants upon helper plasmid-mediated transfer to a recipient strain of Bacteroides distasonis . To improve the potential of pKBF367-1 (14.7 kb) as cloning vector, successive deletions generated derivatives of 12.8, 10.5 and 9.3 kb, which were still able to replicate in B. distasonis 419. These bifunctional vectors were successfully employed to introduce transposon Tn 501 (Hg^r) into B. distasonis 419, but expression of mercury resistance was not observed. This plasmid vehicles series may be useful for cloning Bacteroides genes in E. coli and studying their expression in a heterologous Bacteroides strain.