Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194

Summary We have constructed a hybrid plasmid, pBC1, which consists of plasmid pC194 with an insert of B. subtilis DNA at its HindIII restriction site. This plasmid is stably maintained in B. subtilis. In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation. Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficieny., as defined by recE in the recipient cell. The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size. A model of DNA proccssing during plasmid transformation of B. subtilis is presented.     

Cloning and expression of bacteriophage SP02 DNAZ polymerase gene L in Bacillus subtilis, using the Staphylococcus aureus plasmid pC194

HindIII restriction endonuclease fragments of DNA from temperate Bacillus subtilis bacteriophage SP02 were cloned in B. subtilis by using the plasmid pC194. Three hybrid plasmids which permit growth of the mutant SP02 susL244 in suppressor-negative bacteria were isolated. SP02 gene L is thought to code for a DNA polymerase essential for autonomous replication of SP02 DNA. Extracts of bacteria carrying one of these hybrid plasmids, pC194-96, had 10- to 30-fold increased DNA polymerase activity. The plasmid-induced DNA polymerase activity differed from that of the known B. subtilis DNA polymerases in several respects. The results of the experiments support the idea that phage SP02 codes for a new DNA polymerase.     

Effect of the pC194 plasmid on the development of Bacillus thuringiensis phages

Bacillus thuringiensis var. galleriae strains were transformed by plasmid pC194, coding for chloramphenicol resistance (CmR). Efficiency of plating and the yields of bacteriophages Tg13 and Tg27 maturating in CmR transformant cells were decreased for 2-3 orders as compared with the ones in parental strains. The CmR transformants are characterized by the increased level of spontaneous induction of bacteriophage Tg22.     

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.Non-standard abbreviations BSA bovine serum albumin - CAT chloramphenicol acetyltransferase - Cm^R chloramphenicol resistant - PAB Penassay broth - SDS sodiumdodecylsulfate - Tc^R tetracycline resistant     

Rolling circle replication of single-stranded DNA plasmid pC194.

A group of small Staphylococcus aureus/Bacillus subtilis plasmids was recently found to replicate via a circular single-stranded DNA intermediate (te Riele et al., 1986a). We show here that a 55 bp region of one such plasmid, pC194, has origin activity when complemented in trans by the plasmid replication protein. This region contains two palindromes, 5 and 14 bp long, and a site nicked by the replication protein. DNA synthesis presumably initiated at the nick in the replication origin can be terminated at an 18 bp sequence homologous to the site of initiation, deriving from another plasmid, pUB110, or synthesized in vitro. This result suggests that, similar to the Escherichia coli single-stranded DNA phages, pC194 replicates as a rolling circle. Interestingly, there is homology between replication origins and replication proteins of pC194 and the phage phi mX174.     

Replication origin of a single-stranded DNA plasmid pC194.

The replication of the single-stranded (ss) DNA plasmid pC194 by the rolling circle mechanism was investigated using chimeric plasmids that possess two pC194 replication origins. One of the origins was intact, whereas the other was either intact or mutated. The origins were activated by inducing synthesis of the pC194 replication protein, under the control of lambda phage pL promoter. Initiation of pC194 replication at one origin and termination at the other generated circular ssDNA molecules smaller than the parental chimeric plasmid. From the nature and the amount of ssDNA circles, the activity of an origin could be assessed. Our results show that (i) the signal for initiation of pC194 replication is more stringent than that for termination; (ii) the sequence and structure of the origin are important for its activity and (iii) successful termination of one replication cycle is not followed by reinitiation of another. This last observation differentiates a ssDNA plasmid (pC194) from a ssDNA phage (phi X174).     

Imprecise excision of plasmid pE194 from the chromosomes of Bacillus subtilis pE194 insertion strains.

Plasmid pE194 has been shown to be rescued by integration after cultivation of infected Bacillus subtilis recE4 cells at a restrictive high temperature. The plasmid is also spontaneously excised from the chromosome at a low frequency by precise or imprecise excision (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). We have investigated nine excision plasmids, carrying insert DNA 1 to 6 kbp in length, either in a complete pE194 or in a partially deleted pE194 copy. Type 1 (additive) excision plasmids have the left- and right-junction DNAs preserved as 13-bp direct repeats (5'-GGGGAGAAAACAT-3') corresponding to the region between positions 864 and 876 in pE194. In type 2 (substitutive) excision plasmids, a conserved 13-bp sequence remains only at the right junction while the left junction has been deleted during the excision process. The type 3 excision plasmid carries at each junction the tetranucleotide 5'-TCCC-3', present in pE194 between positions 1995 and 1998. Although we isolated the excision plasmids from different integration mutants, the insert DNAs of eight independently isolated plasmids showed striking sequence homology, suggesting that they originated from one distinct region of the B. subtilis chromosome. Thus, we postulate that imprecise excision of pE194 occurs most frequently after its translocation from the original insertion site into a preferred excision site within the host chromosome. The imprecise excision from this site occurs at excision breakpoints outside the pE194-chromosome junctions in a chromosomal region which remains to be investigated further.     

Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis.

pE194, a 3.5-kilobase multicopy plasmid, confers resistance to the macrolide-lincosamide-streptogramin B antibiotics in Bacillus subtilis. By molecular cloning and deletion analysis we have identified a replication segment on the physical map of this plasmid, which consists of about 900 to 1,000 base pairs. This segment contains the replication origin. It also specifies a trans-acting function (rep) required for the stable replication of pE194 and a negatively acting copy control function which is the product of the cop gene. The target sites for the rep and cop gene products are also within this region. Two incompatibility determinants have been mapped on the pE194 genome and their properties are described. One (incA) resides within the replication region and may be identical to cop. incB, not located in the replication region, expresses incompatibility toward a copy control mutant (cop-6) but not toward the wild-type replicon.     

Generation of deletions through a cis-acting mutation in plasmid pC194

Summary When plasmid pC194-1 is ligated to pBR322 to generate plasmid pHV15-1, deletions occur with high frequency within the joined pBR322 DNA. Generation of deletions is recE4 independent, and occurs in B. subtilis with a 1,000-fold higher frequency than in Escherichia coli. In the hybrid plasmid pVH15-1, deletion end-points are not at random, but at defined locations within pBR322. We propose that the base alteration, characterizing pC194-1, has stabilized within the plasmid a stem/loop structure, which acts as a deletion generator.     

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.     

Study of CAT gene expression in Sa and pC194 plasmids in Escherichia coli, Francisella tularensis, and Bacillus subtilis cells]

The unit activities were defined for chloramphenicol-acetyltransferases coded for by the cat-genes of the plasmids Sa and pC194 in Francisella tularensis, Escherichia coli and Bacillus subtilis cells. Francisella tularensis cells were shown to hold intermediate position between Escherichia coli and Bacillus subtilis cells in their ability to express the genes of the different taxonomic origin. The direct dependence was found between the dose of the gene coding for chloramphenicol-acetyltransferase synthesis and efficiency of the gene expression, minimal inhibiting concentration of the antibiotic and colony size on the media containing chloramphenicol.     

Isolation and complementation of temperature-sensitive replication mutants of Staphylococcus aureus plasmid pC194

Temperature-sensitive replication (Tsr) mutants have been isolated from the Staphylococcus aureus plasmid pC194. For three of the four mutant plasmids tested (pSAO801, pSAO802, and pSAO804) the segregation kinetics suggested a complete block of plasmid replication at 43 degrees C. The replication defects of three mutant plasmids: pSAO802, pSAO803, and pSAO804 could be complemented by recombinant plasmids carrying a segment from either the wild type or the other mutant, pSAO801. There was no complementation when the segment carried by the recombinant plasmid was derived from one of the three complementable mutants. These data were taken as evidence for the involvement of a diffusible, plasmid-encoded product, RepH, in pC194 replication. The complementation of the fourth Tsr mutant, pSAO801, could not be tested due to an abnormal susceptibility of this mutant to the incompatibility expressed by recombinants carrying segments derived from pC194 or its mutants. A single mutation was found to be responsible for both pSAO801 instability and its altered incompatibility properties but the nature of the defect has not yet been elucidated.     

Interference of plasmid pCM194 with lysogeny of bacteriophage SP02 in Bacillus subtilis.

Three observations indicated that the 2-megadalton chloramphenicol resistance plasmid pCM194 interferes with SP02 lysogeny of Bacillus subtilis. SP02 plaques formed on B. subtilis(pCM194) appeared almost clear, whereas plaques produced on plasmid-free or pUB110-containing cells contained large turbid centers. The number of phages spontaneously liberated by B. subtilis(SP02) was increased 10-fold or more when pCM194 was also present in the lysogens. Lastly, growth of B. subtilis(SP02, pCM194) for approximately 20 to 25 generations resulted in essentially complete loss of the prophage. This interference was not observed with pUB110 or pE194, and the pCM194 interference was not directed against B. subtilis temperate phage phi 105, which is unrelated to SP02. Lytic replication of SP02 appeared to be unaffected by pCM194. pCM194 interference with SP02 lysogeny was demonstrable in recombination-proficient strains and a recE mutant of B. subtilis. SP02 prophage which were noninducible due to the phage ind mutation were resistant to pCM194 interference. pCM194 interference was lost when the entire pCM194 molecule was joined at its unique HpaII site or at one of the two MboI sites to pUB110 or pUB110 derivatives. pBR322 joined to pCM194 at the same MboI site or at the HindIII site produced chimeras that retained the ability to interfere with SP02 lysogeny. A three-part plasmid constructed by joining pBR322 to pCM194 (at HindIII sites) and to pE194 (at PstI sites) was compatible with the SP02 prophage and showed a temperature-sensitive replication phenotype characteristic of the pE194 replicon. One explanation for the interference involves competition for a host component between an SP02 genome attempting to establish lysogeny and plasmids whose replication is directed by the pCM194 replicon.     

Identification of plasmid and Bacillus subtilis chromosomal recombination sites used for pE194 integration.

The plasmid pE194 (3.7 kilobases) is capable of integrating into the genome of the bacterial host Bacillus subtilis in the absence of the major homology-dependent RecE recombination system. Multiple recombination sites have been identified on both the B. subtilis chromosome and pE194 (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). The B. subtilis chromosomal recombination sites were recovered by genetic cloning, and these sites were studied by nucleotide sequence analysis. Recombination had occurred between regions of short nucleotide homology (6 to 14 base pairs) as indicated by comparison of the plasmid and the host chromosome recombination sites with the crossover sites of the integration products. Recombination between the homologous sequences of the plasmid and the B. subtilis genome produced an integrated pE194 molecule which was bounded by direct repeats of the short homology. These results suggest a recombination model involving a conservative, reciprocal strand exchange between the two recombination sites. A preferred plasmid recombination site was found to occur within a 70-base-pair region which contains a GC-rich dyad symmetry element. Five of seven pE194-integrated strains analyzed had been produced by recombination at different locations within this 70-base-pair interval, located between positions 860 and 930 in pE194. On the basis of these data, mechanisms are discussed to explain the recombinational integration of pE194.