Characterization of a macrolide, lincosamide, and streptogramin resistance plasmid in Staphylococcus epidermidis.

A strain of Staphylococcus epidermidis was transduced to erythromycin resistance, and all of the transductants exhibited the macrolide, lincosamide, streptogramin B resistance phenotype. Curing and antibiotic disk studies also indicated that these resistances were controlled by a single plasmid determinant and were constitutive. Agarose gel electrophoresis of plasmid deoxyribonucleic acid (DNA) from donor, cured, and transduced strains showed that a single plasmid was responsible. This plasmid, designated pNE131, was examined for sequence homology to two other plasmids, pE194 and p1258, from Staphylococcus aureus, which also code for erythromycin resistance. DNA from plasmids pNE131 and pE194 hybridized with one another, but no extensive homology to pI258 with either pNE131 or pE194 was found. Restriction endonuclease digests of pNE131 and pE194 showed no common fragments. However, sequence homology was localized to the nucleotides in pE194 that code for the 29,000-dalton protein responsible for erythromycin resistance. pNE131 was calculated to have 2,220 base pairs and is the smallest naturally occurring plasmid with a known function yet reported in S. epidermidis.     

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.     

Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells.

The minicell system of Bacillus subtilis has been used to study the expression of plasmid genes using several R plasmids derived from Staphylococcus aureus. pE194, pC194, and pUB110 as well as several mutant and in vitro recombinant derivatives of these plasmids segregate into minicells. A copy control mutant of pE194 was used to show that the extent of segregation is proportional to the copy number. The polypeptides specified by these plasmids were examined by SDS-polyacrylamide gel electrophoresis. Six proteins specified by pE194, an erythromycin resistance plasmid, were identified using cop mutants. These comprise about 90% of the potential coding capacity of the 2.4-Mdal pE194 plasmid. One of these proteins (29,000 daltons) is inducible by erythromycin in the wild type pE194 but is synthesized constitutively in a mutant derivative which also expresses antibiotic resistance constitutively. Several other proteins are detected only in copy control mutants. pUB110, a kanamycin resistance plasmid, expresses three major proteins which comprise 50% of the coding capacity of this 3.0-Mdal plasmid. Two additional minor proteins are occasionally observed. pC194 (2.0 Mdal), which confers chloramphenicol resistance, expresses two polypeptides comprising about 25% of its coding capacity. One of these polypeptides (22,000 daltons) is inducible by chloramphenicol. pBD9, an in vitro composite of pUB110 and pE194, probably expresses all of the major parental plasmid proteins with the exception of one from pUB110 and one from pE194.     

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.     

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.     

Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome.

The plasmid pE194 is unable to replicate in Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). When linked to resident bacteriophage sequences, pE194 was able to integrate into the L. lactis subsp. lactis chromosome either by Campbell-like recombination or by double crossing over with deletion. Integration occurred into the DNA of the prophage and prevented its multiplication. When a selective pressure was applied to an integrant in which pE194 was flanked by two direct repeats of prophage fragment, amplification of pE194 and the prophage fragment was observed. The pE194 copy number was assessed at six to nine, and amplification was stable upon growth under nonselective conditions.     

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.     

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.     

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.     

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.     

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 replication stimulates DNA recombination in Bacillus subtilis

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.     

Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.     

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.     

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.     

Sequence rearrangements in the plasmid ColV,I-K94

ColV,I-K94 is a conjugative (F-like) plasmid specifying colicins V and I. Numerous rearrangements were observed in ColV,I-K94 plasmids from three culture collections and in derivatives which specified larger inhibition zones and increased titers of colicin V. Several of the rearrangements in ColV,I-K94 derivatives specifying higher titers of colicin V involved a 1.3 ± 0.08-kb inverted repeat sequence which was separated by a 3.3 ± 0.2-kb sequence. This was rearranged in one derivative, pKH46, such that the 3.3-kb sequence became flanked by a 2.5 ± 0.15-kb inverted repeat which was subsequently altered to form a 2.1 ± 0.1-kb inverted repeat. In two other derivatives, the 3.3-kb sequence was deleted together with all of the flanking inverted repeat (in pKH39-2) or part of each repeat (in pKH39-1). A 0.5 ± 0.1-kb sequence adjacent to one of the 1.3-kb repeats was inverted or substituted in both of these plasmids. A novel 12.9 ± 0.7-kb inverted duplication separated by a 7.3 ± 0.4-kb sequence was present in the ColV,I-K94 derivative, pKH41, and insertion of the γδ element was detected in another derivative, pKH46-1, after this nonconjugative plasmid had been mobilized by the F plasmid. The genes for colicin V and for immunity to colicin V were mapped to a 3.6-kb sequence. The copy numbers of both pKH39-2 and pKH46-1::γδ were greater than those of pKH38 from which they were derived.     

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.