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.     

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.     

Staphylococcal plasmid cointegrates are formed by host- and phage-mediated general rec systems that act on short regions of homology

Summary Cointegrates involving pairs of compatible staphylococcal plasmids can be isolated either by co-selection during transduction (Novick et al. 1981) or by selection for survival at the restrictive temperature of a thermosensitive, replication defective plasmid in the presence of a stable one. Cointegrates are formed by recombination at two specific sites, RS_A and RS_B. RS_B is present on each of six plasmids analyzed, namely pT181, pE194, pC194, pS194, pUB110, and pSN2, and RS_A is present on two of these, pT181 and pE194. In this communication, it is shown that the RS represent short regions of homology (RS_A is some 70 bp in length and RS_B is about 30) embedded in largely non-homologous contexts and that the crossovers take place within these homologous regions. The pT181 and pE194 RS_A sequences contain several mismatches which permit the localization of the crossover events to several different sites within the overall RS segment. The recombination system involved is therefore general (homology-specific) rather than site-specific (sequence-specific). Mismatches included within the crossover region are always corrected to the pT181 configuration. The cointegrates are therefore formed by a relatively efficient general rec system that recognizes short regions of homology and gives rise to Holliday junctions that probably involve very short heteroduplex overlaps. The sequence results are consistent with asymmetric single-strand invasion of a contralateral gap with nucleotide conversion by copying. It is noted that RS_B has substantial homology with the par sequence of plasmid pSC101, suggesting that it may be involved in plasmid partitioning.     

Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

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.     

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.     

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.     

N-Methyl transferase of Streptomyces erythraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci

The nucleotide sequence of a structural gene ermE for ribosomal RNA (rRNA) N6-amino adenine N-methyl transferase (NMT) of Streptomyces erythraeus, cloned by Thompson et al. [Gene 20 (1982) 51-62], has been determined. The NMT amino acid (aa) sequence deduced from the nucleotide sequence contains extensive homology to aa sequences of cognate NMTs specified by: (1) plasmid pE194 from Staphylococcus aureus, 30% G + C, ermC; (2) plasmid pAM77 from Streptococcus sanguis, 43% G + C; as well as to (3) a chromosomal determinant from Bacillus licheniformis 759, 46% G + C, ermD, cloned in a recombinant plasmid pBD90. These findings suggest that all four NMT structural genes could have evolved from a common progenitor sequence despite the wide range of % G + C of the erm genes reflecting their current respective hosts. Comparison of the four NMT sequences with respect to localized hydrophobicity averaged over a moving window of 11 aa indicates that the common features of localized hydrophobicity that characterize the C-terminal portion of the ermE and ermD proteins are distinguishable from a contrasting pattern of hydrophobicity that characterizes the ermC and pAM77-coded proteins.     

Region of the streptococcal plasmid pMV158 required for conjugative mobilization.

The nonconjugative streptococcal plasmid pMV158 can be mobilized by the conjugative streptococcal plasmid pIP501. We determined the sequence of the 1.1-kilobase EcoRI fragment of pMV158 to complete the DNA sequence of the plasmid. We showed that an open reading frame, mob (able to encode a polypeptide of 58,020 daltons), is required for mobilization of pMV158. An intergenic region present in the EcoRI fragment contains four lengthy palindromes that are found also in one or more of the staphylococcal plasmids pT181, pE194, and pUB110. One palindromic sequence, palD, which is common to all four plasmids, also appeared to be necessary for mobilization. Circumstantial evidence indicates that this sequence contains both an oriT site and the mob promoter. The Mob protein is homologous in its amino-terminal half to Pre proteins encoded by pT181 and pE194 that were shown by others to be essential for site-specific cointegrative plasmid recombination; their main biological function may be plasmid mobilization.     

The nucleotide sequence of Staphylococcus aureus plasmid pT48 conferring inducible macrolide-lincosamide-streptogramin B resistance and comparison with similar plasmids expressing constitutive resistance

The complete nucleotide sequence of a naturally occurring Staphylococcus aureus plasmid, pT48 (from S. aureus strain T48), has been determined. The 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin B (MLS) type antibiotics. It is similar to the constitutive MLS resistance plasmid, pNE131, from Staphylococcus epidermidis and shows homology with S. aureus plasmids pSN2 and pE194. It contains a palA structure homologous to that on S. aureus plasmid pT181. The open reading frame, ORF B, within the pSN2 homologous region has a frameshifted C-terminus, relative to pNE131, resulting in a smaller, 158 amino acid putative polypeptide. The pE194 homologous region has the ermC resistance determinant and retains the leader region, deleted in pNE131, required for inducible expression of an adenine methylase. Another naturally occurring S. aureus strain, J74, shows constitutive resistance to erythromycin and contains a small plasmid, pJ74, which is similar to pNE131 but with a different deletion in the leader sequence. The results are consistent with the translational attenuation model for ermC expression.     

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.     

A site-specific recombination function in Staphylococcus aureus plasmids.

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.     

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.     

Similarity of minus origins of replication and flanking open reading frames of plasmids pUB110, pTB913 and pMV158.

Plasmids pMV158 and pTB913, originating from Streptococcus agalactiae and a thermophilic Bacillus respectively, were sequenced to completion. Both contained a BA3-type minus origin of replication and an RSA-site, believed to constitute a site-specific recombination site. These two regions were more than 99% homologous to the corresponding regions of the Staphylococcus aureus plasmid pUB110. Deleting the BA3-type minus origin resulted in the accumulation of a considerable amount of single-stranded DNA, both in L. lactis subsp. lactis and B. subtilis, indicating that this minus origin was functional in both bacterial species. Like pUB110, both plasmids contained an open reading frame encoding a putative plasmid recombination enzyme (Pre protein), which was located downstream of the RSA-site. On the basis of sequence comparisons between pUB110, pMV158, pTB913, pT181, pE194, pNE131 and pT48 two distinct families of RSA-sites and Pre proteins could be distinguished.     

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.     

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.     

An in vitro coupled transcription-translation system from Staphylococcus aureus

We have developed a Staphylococcus aureus cell-free system that is capable of directing DNA-dependent synthesis of proteins. The staphylococcal plasmids pE194 and pSK265 were used to characterize this system. The in vitro system was found to direct the synthesis of the appropriate proteins predicted from the nucleotide sequence of the plasmids. As is the case in vivo, low levels of the inducer, erythromycin, promoted the synthesis of the pE194-encoded ribosomal RNA methyltransferase in the in vitro system.     

RecE-independent recombination of plasmids in Bacillus subtilis cells

The pUB110 and pE194 plasmid cointegrates have been isolated and examined in rec+ and recE4 strains of Bacillus subtilis. Cointegrates were shown to be formed by recombination at the specific site present on both parental plasmids as a short region of homology designated RSA. The RSA consists of 63 nucleotides in pE194 and 49 in pUB110; the length of its fully conserved core segment is 10 nucleotides. All cointegrates examined were formed by single crossover event taking place within the core segment, and as a result they have identical nucleotide sequences of recombination junctions. No conversion of mismatched base pairs to nucleotide sequences originally belonging to one of the parental plasmids was found. Though the action of RecE gene did not affect the frequency of cointegrate formation, it was reduced in rec149 host by one order of magnitude. Cointegrates retained their stability during transformation.     

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.