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.     

Integration of various plasmids into the Bacillus subtilis chromosome

Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied. Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome. The sites of plasmids for illegitimate recombination were determined. It was shown that the integration into the Bac. subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid. The influence of different Bac. subtilis rec mutations on the frequency of integration was studied.     

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.     

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.     

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.     

Organization of the pE194 genome

Summary pE194 is a 3.5 kilobase erythromycin resistance plasmid which was originally isolated from Staphylococcus aureus and has been introduced into Bacillus subtilis. This plasmid specifies at least five polypeptides, detectable in minicell extracts, one of which (E3) is inducible by erythromycin and is required for the expression of erythromycin resistance. We have constructed a detailed restriction endonuclease cleavage site map of pE194, and have localized the DNA sequences which code for the five polypeptides on the map. Four of the five polypeptides (E2, 3, 4 and 5) are specified by a region of the genome which has half the coding capacity required if these proteins were specified by contiguous genes. The determinant of E3 inducibility is located in the same segment. Based on the deficiency in coding capacity and on additional evidence including peptide mapping, we suggest that either the genes for these four polypeptides overlap, and are read in the same frame, or that some of these proteins represent degradation products. The latter alternative appears less likely since E3 is regulated independently of the other three proteins. The fifth protein (E1) is probably transcribed in the opposite direction. Strand separation and hybridization experiments confirm that both strands are transcribed. Hybridization of labeled RNA from a plasmid-carrying strain to restriction fragments of pE194 reveals that the expected plasmid sequences are transcribed in vivo, as is a region of the genome which is near the replication origin and which does not specify any known polypeptide. The map locations of 3–4 RNA polymerase binding sites are presented. Two of them are found in the segm ent which is predicted to contain the E1 and the E2-5 promoters, and a third is in the region of the replication origin.     

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.     

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.     

Interplasmid illegitimate recombination in Bacillus subtilis cells

The illegitimate recombination between S. aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E. coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B. subtilis was studied. Plasmid cointegrates were generated with the frequency of 1-3.10(-8). Among the 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.). One of the recombinants resulted from nonhomologous recombination. The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B. subtilis chromosome (Bashkirov et al. 1987) was demonstrated. Possible mechanisms of illegitimate recombination are discussed.     

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.     

Integration of hybrid plasmids-derivatives of staphylococcal plasmid pE3692 and pBR322 vector with Bacillus subtilis chromosome

Bacillus subtilis is a nonpathogenic microorganism which certainly for also that feature is widely used in several biotechnological processes. Certainly, because of that an elaboration of gene cloning methods in Bacillus subtilis cells is of great significance both from scientific and practical point of view. It was found hybrid plasmids constructed by ligation of pE3692 and pE3692-9 staphylococcal plasmids with pBR322 vector can be integrated with B. subtilis chromosome. Selection of cells in the presence of erythromycin at 50 degrees C allows to isolate clones containing exclusively integrated plasmids. Growth rate of cells containing these plasmids in the selective medium is the same or slightly higher at 50 degrees C than at 37 degrees C. It seems that hybrid plasmid tested pBE4 and pBE91 after appropriate reconstruction in vitro could serve as vectors for B. subtilis gene cloning.     

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.     

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.     

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.     

Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies.

pE194 is a small plasmid (isolated originally in Staphylococcus aureus) which confers erythromycin-inducible resistance to macrolide, lincosamide, and streptogramin type B (MLS) antibiotics. The nucleotide sequence of pE194 contains 3,728 base pairs (bp), corresponding to a molecular mass of 2.4 million daltons. By means of site-specific cleavage with restriction endonucleases and cloning resultant fragments, determinants of the two major biological functions of p E194, i.e., inducible MLS resistance and replication, could be localized and assigned to specific sequences in the plasmid. Restriction endonuclease TaqI cut pE194 at three sites. TaqI fragment A (1,443 bp) contained the determinant for inducible MLS resistance, whereas TaqI fragment B (1,354 bp) contained a determinant necessary for plasmid replication. Regulatory mutations resulting in constitutive expression of MLS resistance mapped in TaqI fragment A, whereas a mutation associated with elevated plasmid copy number was mapped in TaqI fragment B. Also mapping in TaqI fragment B was a plasmid replication determinant comprising two sets of inverted complementary repeat sequences, one of which spanned 124 bp and was adjacent to a second smaller set which was rich in guanine and cytosine residues. pE194 contained six open reading frames which were theoretically capable of coding for proteins with maximum molecular masses as follows (in daltons): A, 48,300; B, 29,200; C, 14,000; D, 13,900; E, 12,600; and F, 2,700. Insertion of plasmid pBR322 into the single PstI site located in frame A of pE194 resulted in a composite plasmid which could replicate in both Bacillus subtilis and Escherichia coli, suggesting that an intact polypeptide A is dispensable for both replication of pE194 and for MLS resistance. Frame B specified inducible MLS resistance, whereas frame F specified the putative peptide associated with the proposed B determinant translational attenuator. The extent to which frames C, D, and E, all contained in TaqI fragment B, were translated into polypeptide products is not known; however, a base change in frame E was found in a comparison between the high-copy-number mutant, cop-6, and the wild-type strains.     

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.     

Yeast centromeric plasmids as shuttle vectors between Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae

A number of hybrid plasmids which can autonomously replicate in E. coli, B. subtilis and S. cerevisiae was constructed. Replication of these plasmids both in yeast and in B. subtilis starts on a sequences originating from Staphylococcus aureus plasmids pC194 and pE194. In yeast these hybrids are unstable like those yeast vectors which contain eukaryotic ARSs, but their stability has been increased by addition of yeast centromeric sequence. Both pC194 and pE194 DNAs contain sequences which reveal strong similarities with the yeast ARS consensus. Nevertheless the replication efficiences of these plasmids in yeast are different.     

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein.

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.