Nucleotide sequence of the constitutive macrolide-lincosamide-streptogramin B resistance plasmid pNE131 from Staphylococcus epidermidis and homologies with Staphylococcus aureus plasmids pE194 and pSN2.

The complete nucleotide sequence of the Staphylococcus epidermidis plasmid pNE131 is presented. The plasmid is 2,355 base pairs long and contains two major open reading frames. A comparison of the pNE131 DNA sequence with the published DNA sequences of five Staphylococcus aureus plasmids revealed strong regional homologies with two of them, pE194 and pSN2. The region of pNE131 containing the reading frame which encodes the constitutive ermM gene is almost identical to the inducible ermC gene region of pE194, except for a 107-base-pair deletion which removes the mRNA leader sequence required for inducible expression. A second region of pNE131 contains an open reading frame with homology to the small cryptic plasmid pSN2 and potentially encodes a 162-amino-acid protein.     

Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194.

The complete nucleotide sequence of pADB201, a 1.7-kilobase cryptic plasmid from Mycoplasma mycoides subsp. mycoides, is reported. The sequence contains a single large open reading frame capable of coding for a polypeptide of up to 198 codons long. The sequence of the putative polypeptide shows significant similarity to that of the repF gene product of staphylococcal plasmid pE194.     

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.     

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).     

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.     

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.     

Origin and mode of replication of plasmids pE194 and pUB110

Replication of the multicopy antibiotic resistance plasmids pE194 and pUB110 has been studied in Bacillus subtilis. Based on electron microscopic analysis of replication intermediates and on evidence from deletants, we have determined that both plasmids replicate unidirectionally from fixed origins by a Θ mechanism. The location of these origins and the directions of replication have been determined relative to the physical maps. A cointegrate of pE194 and pUB110 has also been studied and evidence is presented from electron microscopy, temperature resistance, and copy number analysis that the cointegrate uses both parental replication origins.     

Replication in yeasts of plasmid pE194 from Staphylococcus aureus

The ability of the plasmid pE194 from S. aureus to serve as an autonomously replicating sequence (ARS) in yeast was shown. The hybrid plasmid pLD744 that contains pE194 and the yeast LEU2 gene sequences is unstable in yeast like other YRp-vectors: the mitotic stability of the pLD744 was as much as 1%. The plasmid pLD712 that differs from pLD744 by the existence of a centromeric sequence from the chromosome III of yeast Saccharomyces cerevisiae reveals about one order greater stability. The observation that there are some sequences in the primary structure of the pE194 which strongly conform to the ARS consensus in yeast inclines us to infer that the existence of ARS consensus on pE194 DNA is not sufficient for its effective replication in yeast.     

Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids.

The 2,647-bp nucleotide sequence of cryptic plasmid pTD1, isolated from the oral spirochete Treponema denticola, was determined. The sequence revealed two open reading frames, A and B, which encode polypeptides of 335 and 235 amino acids, respectively. Open reading frame A shows sequence similarity to genes that encode replication proteins from a group of plasmids common in gram-positive bacteria, which replicate via a single-stranded intermediate.     

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.     

Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.

A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.     

Restriction map of an agropine-type Ri plasmid and its homologies with Ti plasmids

The Ri plasmid of an agropine-type Agrobacterium rhizogenes, strain HRI, was cloned in a cosmid and mapped with the restriction endonucleases BamHI, EcoRI, KpnI, SmaI, and XbaI. This plasmid is almost identical to pRi1855 and pRiA₄b. A study by Southern hybridizations of the homologies with octopine pTiB₆806 and nopaline pTiC58 makes it possible to propose the localization of certain functions on this plasmid, such as virulence, agropine catabolism, agropine synthesis, and the origin of replication.     

Transient generation of displaced single-stranded DNA during nick translation

We show that displaced single-stranded overhangs are transiently generated and destroyed during nick translation by E. coli DNA polymerase I. Evidence that hyper-rec mutants have an increased frequency of such overhang structures is discussed. The transient generation of overhangs may be significant for general recombination. The 5' leads to 3' exonuclease activity of polymerase I specifically hydrolyzes such overhangs to yield a nick. Overhangs are generated by polymerization, but after every polymerization step, either polymerase or exonuclease can act--55% of the time, polymerization occurred first. At this frequency overhangs of greater than or equal to 12 nucleotides are generated every 1300 nucleotides polymerized. We suggest that many DNA strand discontinuities are displaced single-stranded overhangs, rather than gaps or simple nicks.     

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.     

Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.