Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli.

Summary (1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, an in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.     

Plasmid transformation in Bacillus subtilis. The significance of partial homology between plasmid and recipient cell DNAs

Summary A series of hybrid plasmids consisting of pC194 or pUB112 and B. subtilis DNA were constructed. In contrast to plasmid pC194, purified monomeric forms of such plasmids were active in transformation, provided the recipient cells were recombination proficient. Similarly the monomers of PC194 derived plasmids, containing bacteriophage 105 DNA were able to transform 105 lysogenic but not nonylsogenic cells. From the results it is concluded that the presence of DNA/DNA homology between chromosomal DNA of the recipient cell and part of the hybrid plasmids used is a sufficient condition to endow monomeric plasmids with transforming activity.This work is part of the Doctoral Thesis to be submitted by A. Iglesias to the Freic Universität Berlin     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).     

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.     

Generation of transducing particles in Staphylococcus aureus.

Transduction of plasmid pC194 and bacteriophage phi 11de varied inversely with the multiplicity of infection. As the multiplicity of infection decreased from 10(-1) to 10(-5) PFU/CFU, the transduction frequency of pC194 increased 10(4)-fold; the transduction frequency of phi 11de increased 300-fold with a 100-fold decrease in multiplicity of infection. Physical and genetic analysis of the transduced DNA showed that pC194 resided in the phage particle as a random, circularly permuted linear concatemer. In DNA prepared from phage that cotransduced pC194 and phi 11de, pC194 resided in the transducing phage primarily as a linear multimer of 15.8 kilobases, or about 5.4 pC194 monomers. The pC194 multimer was randomly inserted into the phi 11 genome.     

Expression of α-amylase gene from Bacillus stearothermophilus in Iactobacillus sanfrancisco

Summary pC 194Amy, a construct containing an amylase encoding gene, was introduced in Lactobacillus sanfrancisco CB1 by electroporation and the Amy gene was expressed. Amylase activity was extracellular and retained after 140 generations. The growth of the transformant with 10 g starch/L and 5 g maltose/L was similar to that of the wild type in 10 g maltose/L. L. sanfrancisco CB1 transformant harboured pC 194Amy, a small DNA fragment and did not possess the native plasmid. The small DNA fragment was demonstrated to be a deletion product of pC194Amy containing the Amy sequence. L. sanfrancisco CB1 was also transformed with pGKV210, pNZ12 and pMG36e by electroporation.     

Generation of linear multigenome-length plasmid molecules in Bacillus subtilis.

Linear multigenome-length double and single stranded plasmid DNA was identified in a Bacillus subtilis ATP-dependent DNAase mutant strain (addA5) bearing plasmids pC194 or pBD95ts. Plasmid pBC30, a seg mutant of pC194, as well as some pUB110 derivatives with rearrangements external to the minimal replicon, produce high amounts of such a concatemeric DNA, even in Rec+ cells. The synthesis of this type of plasmid DNA occurs in the absence of an active plasmid-encoded Rep protein and is markedly affected in polA5 and recE4 genetic backgrounds. To account for these observations, we propose that the AddAB complex serves to prevent a sigma-type replication of plasmid DNA.     

Plasmid transformation in Bacillus subtilis: Fate of plasmid DNA

Summary Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA, Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA.We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.Some of these results were presented at the 5th European Meeting on Bacterial Transformation and Transfection, September 1980, Florence     

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     

The effect of trimethylpsoralen--crosslinks on entry of donor DNA in transformation and transfection of Bacillus subtilis

Summary Transforming chromosomal DNA, irradiated with long-wave UV light in the presence of 4,5,8-trimethylpsoralen (TMP) binds to competent B. subtilis cells as effectively as non-treated DNA, but its transforming activity is strongly reduced.Uptake studies show that the entry of transforming DNA, after some stimulation by short periods of irradiation in the presence of TMP, decreases proportionally with the dose of irradiation. Crosslinking was quantitated by electron microscopy. Since the number of crosslinks increases proportionally with the dose of irradiation, it is suggested that entry of donor DNA is prevented by crosslinks. The inhibition of entry of DNA is paralleled both by decreased breakdown of crosslinked DNA interacting with competent cells, and decreased breakdown by nuclease activity liberated during protoplasting of competent cultures. These data support the model of Lacks et al. (1976) which postulates that a membrane-bound deoxyribonuclease is engaged in the entry of donor DNA into the competent cell.The transforming activity of the chloramphenicol-resistance carrying plasmid pC194, originally obtained from Staphylococcus aureus, is also destroyed by TMP crosslinks. Contrary to chromosomal DNA, its association with the cells is stimulated by longwave UV irradiation in the presence of TMP, but experiments are presented suggesting that the DNA is still vulnerable to the action of exogenous pancreatic deoxyribonuclease.Transfecting SPP1 DNA is also inactivated by TMP crosslinks. Marker rescue of transfecting DNA containing crosslinks occurs; the extent of rescue of one marker is considerably in excess of that of linked markers.     

A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

A gene controlling segregation of the Bacillus subtilis plasmid pC194

Summary Plasmid pC194-1, a mutant of pC194, and chimeric derivatives of pC194-1 are segregationally unstable in B. subtilis. Such instability could be enhanced by exposure of pC194-1-carrying cells to methyl methanesulfonate. pC194-1 is distinct from pC194 in the addition of two A:T base pairs within the previously defined D region of pC194. Complementation experiments between pC194-1 and other plasmids suggest that the mutation of pC194-1 interferes with the production of a diffusible gene product required for plasmid maintenance.     

Effect of concentration on the subsequent fate of plasmid DNA in human fibroblasts

Summary The physical fate of plasmid DNA after entry into human fibroblasts was studied using Southern hybridisation and electron microscopy. Exposure of the cells (5x10⁵ per well) to pC194 DNA-CaP_i, containing 50 g plasmid DNA, resulted in the occasional formation of interlocked molecules. Exposure to a co-precipitate containing 100 g pC194 plasmid DNA per well resulted in an increase of interlocked molecules by a factor of 10–20 relative to the number of monomers. In addition, new classes of molecules were observed. After prolonged incubation of the cells exposed to the higher DNA concentration, the plasmid DNA was partly contained in structures with a very low electrophoretic mobility. Upon restriction endonuclease digestion of the re-extracted DNA, a pattern of bands was observed, suggesting the involvement of illegitimate recombination between non-random plasmid DNA sequences in the formation of the new classes of molecules.Abbreviations DNA-CaP_i DNA-calcium phosphate co-precipitate - EDTA ethylene-diamino-tetraacetate - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N, N, N, N-tetraacetate - PBS phosphate-buffered saline - PEG polyethylene-glycol - SDS sodium-dodecyl-sulphate - Symbols C1, L1, O1; C2, L2, O2; C3, L3, O3 covalently closed, linear and open circular forms of monomers, dimers and trimers, respectively     

Plasmid maintenance in Bacillus subtilis recombination-deficient mutants

Summary An isogenic set of 11 recombination-deficient mutant strains of Bacillus subtilis has been constructed. Whereas plasmid pUB110 is stably maintained in such Rec^- cells, the high copy number plasmid pC194 is unstable. Instability in Rec^- strains could be mostly attributed to the deleterious effect of the presence of the plasmid on the Rec^- cells' growth capability. In part, instability of pC194 derivatives could also be correlated with the presence of an unusually high amount of multimeric DNA molecules.     

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).     

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.     

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type="Italic">Bacillus</Emphasis> strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.     

Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14

Summary The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepC of pUB110 throughout the protein molecule, but not with RepC of pT181, of R6K or protein RepH encoded by and iniating the replication of pC194.     

Saccharomyces cerevisiae ARS on a plasmid from Staphylococcus aureus

Staphylococcus aureus plasmid pC194 carries three sequences closely related to a consensus sequence defined previously by analysis of different genetic elements which replicate autonomously in yeast Saccharomyces cerevisiae. Two of these enable the plasmid to replicate in yeast, the third does not. A new consensus sequence A/T T T T A T R T T T, 1 bp shorter than the previous one, can be deduced from our results. Replacement of the T with G at the position 9 of the sequence abolishes its activity. The presence of the two active sequences on pC194 genome can be explained by the A + T-rich base composition of the plasmid.