A site-specific recE4-independent intramolecular recombination between Bacillus subtilis and Staphylococcus aureus DNAs in hybrid plasmids

A composite plasmid pLS253 was constructed from pLS103 [carrying the Bacillus subtilis leucine genes on B. subtilis (natto) plasmid pLS28] and pHV14 [a recombinant plasmid composed of pBR322 and the staphylococcal R-plasmid pC194] employing BamHI endonuclease, T4 DNA ligase, and B. subtilis transformation. All the Leu+ Cmr transformants tested harbored not only pLS253 but also two smaller plasmids designated as pLS251 and pLS252. pLS253 DNA, when purified on an agarose gel, retained both Leu+ and Cmr transforming activities; however, in all the Leu+ Cmr transformants, the two smaller plasmids reappeared. pLS251 and pLS252 exhibited Leu+- or Cm4-transforming activity, respectively, and must have been derived from the pLS253 parent by an intramolecular recombination event, since the sum of the pLS251 and pLS252 DNAs represent the entire pLS253 genome. The recombination occurred between specific sites on the B. subtilis (natto) and Staphylococcus aureus plasmids. When the composite plasmid, pLS254, was constructed by BamHI cleavage of pLS251 and pLS252 followed by ligation, Leu+ Cmr transformants segregated two smaller plasmids which were indistinguishable from the original plasmids pLS103 and pHV14, respectively. They must have been derived from pLS254 through a reversal of the original recombination event. No intermolecular recombination between pLS251 and pLS252 DNA was detected. The recombination process was independent of recE function of the host cells, and its mechanism is discussed.     

Site-specific recombination between plasmids of Staphylococcus aureus

Anomalous recombination between two similar but nonidentical, naturally occurring penicillinase plasmids, pI258 and pI524, leading to duplication and deletion of the beta-lactamase locus, is described. Physical mapping of these plasmids by heteroduplex and restriction analysis revealed that the beta-lactamase loci were homologous and in inverted orientation with respect to one another and that their respective locations were separated by a short region of homology. This intervening region of homology included one copy of a segment that was repeated on pI524 in inverted orientation at a distance of 2.2 kilobase pairs and contained a recognition sequence for a site-specific, rec-independent recombination function that caused reversible inversion of this segment on pI524. It is proposed that site-specific, intermolecular recombination involving this repeated sequence was responsible for the observed results.     

A second site-specific restriction endonuclease from Staphylococcus aureus.

A site-specific restriction endonuclease has been isolated from Staphylococcus aureus PS 96. This enzyme, Sau96 I, recognizes the DNA sequence 5'--G-G-N-C-C--3' and cleaves as indicated by the arrows. The enzyme 3'--C-C-N-G-G--5' cleaves adenovirus type 5 and lambda DNA many times, SV40 DNA 10 times and 0X174 RF DNA 2 times. Evidence is presented that the enzyme is involved in biological restriction-modification.     

Relationships between cotransducible plasmids in Staphylococcus aureus.

Cotransduction of two or even three plasmids was observed when a Staphylococcus aureus strain, carrying five distinct, compatible plasmids, was used as donor. An active host recombination system did not seem to be indispensable for plasmid cotransduction, since RecA+ and RecA- donors gave similar cotransduction frequencies. Analysis of plasmids carried by cotransductant clones demonstrated that a genetic interaction can take place between cotransduced plasmids, leading to new plasmids. Some of the properties of these new plasmids are discussed. Another set of experiments tested the ability of a cotransducible plasmid to allow a significant degree of multiplication of a temperature-sensitive plasmid at restrictive temperature. In an attempt to explain the results obtained, a working hypothesis suggesting a transient and reversible association of cotransducible plasmids is presented.     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).     

Penicillinase plasmids of Staphylococcus aureus: restriction-deletion maps.

The derivation of physical-genetic maps of two Staphylococcus aureus penicillinase plasmids—pI258 [28.2 kilobases (kb)] and pII147 (32.6 kb)—is described. The maps are based on data obtained by recombination, deletion, restriction endonuclease digestion, molecular cloning, and insertional inactivation. Evidence is presented for the existence of at least three separate operons transcribed in different directions. Data are presented to show that these plasmids are closely related physically as well as genetically to several others with which they can recombine. Physical mapping studies of one recombinant have helped to pinpoint structural differences between the two parental plasmids.     

Transduction-related cointegrate formation between staphylococcal plasmids: A new type of site-specific recombination

Small R plasmids are frequently cotransduced by staphylococcal transducing phages. Most cotransductants contain independent plasmids indistinguishable from those of the donor strain; occasionally recombinational exchanges can be demonstrated including the formation of stable cointegrates that appear to contain all of the genomes of the two starting plasmids. These cointegrates do not dissociate at a detectable frequency upon subculture of the host strain or upon further transduction. Examination of a series of these cointegrates derived by recombination between various pairs of plasmids has revealed the existence of a new type of site-specific recombination. Nineteen different cointegrates involving five different plasmids were studied by restriction endonuclease analysis and electron microscopic examination of heteroduplexes. To the limits of resolution of these techniques, it can be concluded that each plasmid contains one or more specific sites that is used for the formation of cointegrates with the other plasmids. In addition, the cointegrates are orientation specific as well as site specific so that the recombination process resembles that of prophage integration more than that of transposon insertion. Strikingly, in cases where cointegrates between one plasmid, A, and two or more others (e.g., B and C) have been isolated, the same site on A is used for cointegrate formation with both B and C; moreover, B and C also form cointegrates with each other, using the same site and orientation with which both recombine with A. The formation of cointegrates probably involves identical sequences of ≤ 100 nucleotides and is not demonstrably related to overall homology.     

Mobilization of recombinant plasmids from Staphylococcus aureus into coagulase negative Staphylococcus species.

pC221, a small nonconjugative staphylococcal plasmid, can be mobilized between staphylococci by pG01, a larger conjugative plasmid. pC221 carries the two transacting genes, mobA and mobB, which are needed for its mobilization. The products of these genes create a site-specific single-stranded nick (mobA) and then facilitate DNA transfer (mobB). Several useful Escherichia coli-staphylococcal shuttle plasmids containing the cloned single-stranded nick site were created and successfully mobilized into Staphylococcus aureus and two coagulase-negative staphylococci, S. epidermidis and S. saprophyticus, by providing mob genes (pC221) and conjugative transfer genes (pG01) in trans in the donor. These vectors may offer a genetic system for the introduction of recombinant plasmids into coagulase negative staphylococci.     

Phage-mediated conjugative transfer of plasmids in Staphylococcus aureus]

It was shown possible to transfer nonconjugative plasmids during joint cultivation of the donor and recipient cells by transduction and phage-mediated conjugation. In the latter case it was necessary that the phage in the medium was free and the prophage was present in the recipient cells. Differences in the regularities of the transfer of the nonconjugative plasmids mobilized by the conjugative plasmid or phage were observed.     

Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination

A number of hybrid plasmids, carrying lambda genes involved in site-specific integrative recombination, have been constructed in vitro. Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively. The plasmids RSF2124::lambda-CD, RSF2124::lambda-Cint-c57, and pInt lambda were able to integrate into the chromosome of E.coli at the attB. The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids. Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions. The Southern blotting experiments using [32P]pBR322 DNA and EcoRI fragments of E. coli polA1 chromosome carrying integrated plasmid pInt lambda demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration. In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states. The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells. Only attP and the int gene of lambda are necessary for the integration of hybrid plasmids under conditions of effective int gene expression. If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product. The six-fold decrease of Int protein concentration in the cell (in case of pInt lambda 2 as compared to pInt lambda 1) is critical both for integration and excision.     

Characteristics of penicillinase plasmids from Staphylococcus aureus strains

Penicillinase plasmids are present in most MRSA strains. They are very varying in their genotype and phenotype they confer. Penicillinase plasmids were transduced from 80 hospital MRSA strains to NCTC 8325 and the phenotype as well as the incompatibility group of plasmid were determined. Resistance to cadmium (high and low level), resistance to organic and nonorganic mercury compounds, arsenate/arsenite/antimonium resistance, resistance to bismuth and hypersensitivity to bismuth, resistance to macrolides as well as beta-lactamase production and its inductibility were checked. Among the examined strains 20 different phenotypes of penicillinase plasmids were found. Patterns of penicillinase plasmids were compared to DNA patterns of the investigated strains after digestion with SmaI and separation in pulsed field electrophoresis (PFGE). It was shown that strains with the same PFGE pattern often differ in the type of their penicillinase plasmid. Determining of penicillinase plasmid phenotype could be useful in differentiating S. aureus strains sharing the same pattern of PFGE.     

Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus

Abstract Twelve different chloramphenicol-resistance (Cm^r) plasmids detected in Staphylococcus aureus strains isolated between 1952 and 1981 were characterized by restriction endonuclease, DNA hybridization and heteroduplex analyses. These studies revealed three families of Cm^r plasmids which were distinguished by their chloramphenicol acetyltransferase sequences; the prototype plasmids of the families were pC221, pC223 and pC194. The cat and replication ( rep ) genes of the plasmid pC221 were highly conserved in other pC221 family members and were related to their homologs in the pC223 family plasmids; however, the cat and rep genes of the pC194 family plasmids were distinct.     

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.     

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.     

Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis.

Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUB110, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec- B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 10(4) to 10(5) transformants per microgram of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iord?nescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.