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).     

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.     

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.     

Illegitimate recombination occurs between the replication origin of the plasmid pC194 and a progressing replication fork.

Hybrids between plasmids pC194, pBR322 and the bacteriophage f1 undergo deletions in Escherichia coli. The deletions end most often between nucleotides 1445 and 1446 of pC194. That site probably corresponds to a nick in the replication origin of this plasmid. The localization of the other deletion end appears to be determined by the position of the f1 replication fork. Two models accounting for these data are discussed.     

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA

In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3′-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that a model in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.     

Rolling circle replication of DNA in yeast mitochondria.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.     

Plasmid structural instability associated with pC194 replication functions

The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When gram-positive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S.D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested. It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.     

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.     

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.     

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     

Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids.

Staphylococcus aureus plasmid pE194 manifests a natural thermosensitivity for replication and can be established in several species, both gram positive and gram negative, thus making it attractive for use as a delivery vector. Like most characterized plasmids of gram-positive bacteria, pE194 generates single-stranded DNA. The direction of pE194 replication is clockwise, as determined by the strandedness of free single-stranded DNA. Significant homology exists between a 50-base-pair sequence in the origin of pE194 and sequences present in plasmids pMV158 (Streptococcus agalactiae), pADB201 (Mycoplasma mycoides), and pSH71 (Lactococcus lactis). We used an initiation-termination reaction, in which pE194 initiates replication at its own origin and is induced to terminate at the related pMV158 sequence, to demonstrate that pE194 replicates by a rolling-circle mechanism; the initiation nick site was localized to an 8-base-pair sequence.     

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.     

Synthesis of single-stranded plasmid pT181 DNA in vitro. Initiation and termination of DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

Localization of the replication origin of plasmid pE194.

The pE194 replication origin was localized to a 265-base-pair interval by analyzing the ability of purified pE194 restriction fragments to direct replication of heterologous plasmids. Replication was dependent upon RepF protein supplied in trans. The origin region contained a GC-rich dyad symmetry which may serve as the RepF target.     

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.     

Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage.

Escherichia coli plasmids pBR313 and pBR322 were transduced by phage M13 with low efficiency (10(-8) transductants/phage). Hybrid plasmids pHV12 or pHV33, composed of Staphylococcus aureus plasmid pC194 and pBR313 or pBR322, respectively, were transduced much more efficiently (10(-4) transductants/phage). Inactivation of either of the two zones necessary for pC194 replication, one coding for a protein, the other not, reduced the transforming efficiency of hybrids to the level of pBR322. Activity of the pC194 replication region was not necessary for the formation of chimeras between M13 and the transduced plasmid in the donor cells, but rather for the establishment of the plasmid in the recipient cells.     

Initiation and termination of the bacteriophage phi X174 rolling circle DNA replication in vivo: packaging of plasmid single-stranded DNA into bacteriophage phi X174 coats.

The bacteriophage phi X174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting phi X174 phages. A consequence of this interaction is packaging of single-stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This property was used to study morphogenesis and to analyse the signals for initiation and termination of the rolling circle DNA replication in vivo. It is shown that the size of the DNA had a strong effect on the encapsidation by the phage coats and the infectivity of the particle. Termination was analysed by using plasmids with two phi X (+) origins either in the same orientation or in opposite orientation. Both origins were used with equal frequency. Initiation at one origin resulted in very efficient termination (greater than 96%) at the second origin in the case of two origins in the same orientation. When the two (+) origins have opposite orientations, no correct termination was observed. The second origin in the opposite strand effectively inhibits (greater than 98%) the normal DNA synthesis; i.e. the covalently bound A protein present in the replication fork interacts with the (+) origin sequence in the opposite strand.     

Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5' end of the single pC194 MspI site associated with replication. This 5' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.     

Transformation of Bacillus subtilis by single-stranded plasmid DNA.

The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.