Recombinant plasmids formed in vivo carrying and expressing two incompatibility regions.

The formation in vivo of recombinants between a plasmid of incompatibility group N (R1010-10) and plasmids of groups P (R751) and W (R388) is described. From examination of the molecular weights of these recombinant plasmids, they appear to be cointegrates. These cointegrates have the incompatibility properties of both 'parent' 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.     

Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens.

Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become established only infrequently in recipients with a nopaline Ti plasmid and, vice versa, nopaline Ti plasmids were only rarely established in recipients with an octopine Ti plasmid. Rare clones in which the incoming octopine (nopaline) Ti plasmid had been established despite the presence of a nopaline (octopine) Ti plasmid appeared to harbor cointegrates consisting of the entire incoming Ti plasmid and the entire resident Ti plasmid. The integration event invariably had occurred in a region of the plasmids that is highly conserved in evolution and that is essential for oncogenicity. These results show that octopine and nopaline Ti plasmids cannot be maintained as separate replicons by one and the same cell. Therefore, be definition, these plasmids belong to the same incompatibility group, which has been names inc Rh-1. Agrobacterial non-Ti octopine and nopaline plasmids were found to belong to another incompatibility group. The tumorigenic properties of strains harboring two different Ti plasmids, in a cointegrate structure, were indicative of the virulence genes of both of them being expressed. The agrobacterial non-Ti octopine and nopaline plasmids did not influence the virulence properties encoded by the Ti plasmid.     

RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids.

Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.     

Integration of temperature-sensitive nonconjugative plasmid carrying kanamycin gene into conjugative R plasmids.

A nonconjugative R plasmid, rMS3, whose molecular weight was 2.4 X 10(7) daltons, possessed a kanamycin resistance gene and was thermosensitive in its maintenance in Escherichia coli strains. We mobilized rMS3 with a conjugative R plasmid, R100 or T-tet, and obtained cointegrates carrying all the parental resistance markers. Various markers of the cointegrates were frequently deleted by P1 transduction and the deletion patterns among the different cointegrates were differed from each other. The cointegrates were thermoresistant, but the thermosensitive replicon could be segregated from the thermoresistant cointegrate by deletion. Some cointegrates between rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 into the regulator gene of the transfer loci. The genome size of the cointegrate so far tested was the sum of the sizes of the parental plasmids, indicating that the whole genome of rMS3 could integrate into various sites of the conjugative plasmids R100 and T-tet.     

Polyresistant Enterobacteriaceae strains and their plasmids in a hospital. Medical and theoretical aspects

The properties and origin of multiple resistant strains of Enterobacteriaceae found in the intestine and nasopharynx of infants admitted to the hospital for premature infants were studied. The strains of E. coli of different serovars isolated at various periods contained similar conjugative R plasmids with a molecular weight of 80 Md belonging to the O incompatibility group controlling resistance to kanamycin and physically independent small plasmids controlling resistance to ampicillin (7 Md) and streptomycin-sulfanilamides (4 Md). Multiple drug resistance in the strains of K. pneumoniae was controlled by single large (100-120 Md) plasmid cointegrates with 6-8 resistance markers. Such cointegrates consisted of several potentially independent plasmids, sometimes dividing on transformation of plasmid DNA of the recipient strains of E. coli K12. The small plasmids controlling resistance to ampicillin and streptomycin-sulfanilamides similar to the respective plasmids of E. coli were the constant components of the plasmids cointegrates. The multiple drug resistance in the above strains was combined with high capacity for colonization in premature infants. The medical staff and mothers were the sources of bacterial strains with single plasmids controlling definite types of resistance. It is suggested that the multiple resistant strains of Enterobacteriaceae are formed in hospital as a result of accumulation of the plasmids or plasmid markers and selection. One of the conditions for successive acquisition of new plasmid markers by definite bacterial strains was their high capacity for colonization in patients, which provided constant contacts and genetic exchange of such strains with a wide range of immigrant strains during colonization in the newly admitted patients.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Molecular rearrangements between two plasmids of the FII incompatibility group in different recombination—Deficient Escherichia Coli strains

The frequencies and types of plasmid molecular rearrangements generated in different recombinant mutants which carried two plasmids of the FII incompatibility group were studied. The wild-type cells generated molecular rearrangements mainly by interplasmidic recombination with a frequency of 2.4 x 10(-6) per cell per cell doubling. Cells in which RecF was the principal recombination pathway generated different types of molecular rearrangements that involved either both plasmids or one of the plasmids and the chromosome. The frequencies of molecular rearrangements for these cells were 50-fold greater than those of wild-type cells. The recA- cells, even when the RecE pathway was derepressed, generated rearrangements only between one of the plasmids and the chromosome, at very low frequencies (10(-9]. In wild-type cells and in RecF cells, interplasmidic recombination generated mainly cointegrates carrying DNA deletions. These cointegrates were stable in recA- or recA- RecE+ cells, but unstable in wild-type or RecF+ cells. In the latter, the cointegrates generated smaller plasmids with different molecular structures at relatively low frequencies.     

Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates]

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.     

Mathematical modeling of the evolution of bacterial populations in continuous cultures with regard to the nonmutagenic variability of genome

A mathematical model of the evolution of the genetic structure of the bacterial population during prolonged cultivation in a chemostat has been constructed. In addition to genetic mutations, some factors of the nonmutagenic variability of genome were taken into account, namely, the structural reorganization of plasmid and virus DNA, the DNA loss due to cellular division, the conjugative transfer of plasmids, and the plasmid replication. The general model also takes into account the formation of cellular aggregates during conjugation. The results of numerical and analytical investigation of the special cases of the general model were treated. Simplified mathematical models are considered, which can be used to explain the experimentally observed evolutionary variations resulting from the plurality of evolution attractors, multi-stage microevolutionary transitions, the semi-stable states of the bacterial population genome, and the undamped oscillations of the genetic structure of the population during prolonged cultivation in the chemostat.     

Broad-host-range plasmids from agricultural soils have IncP-1 backbones with diverse accessory genes

Broad-host-range plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to antibiotics and heavy metals or degradation of pollutants. Although some broad-host-range plasmids have been extensively studied, their evolutionary history and genetic diversity remain largely unknown. The goal of this study was to analyze and compare the genomes of 12 broad-host-range plasmids that were previously isolated from Norwegian soils by exogenous plasmid isolation and that encode mercury resistance. Complete nucleotide sequencing followed by phylogenetic analyses based on the relaxase gene traI showed that all the plasmids belong to one of two subgroups (β and ε) of the well-studied incompatibility group IncP-1. A diverse array of accessory genes was found to be involved in resistance to antimicrobials (streptomycin, spectinomycin, and sulfonamides), degradation of herbicides (2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenoxypropionic acid), and a putative new catabolic pathway. Intramolecular transposition of insertion sequences followed by deletion was found to contribute to the diversity of some of these plasmids. The previous observation that the insertion sites of a Tn501-related element are identical in four IncP-1β plasmids (pJP4, pB10, R906, and R772) was further extended to three more IncP-1β plasmids (pAKD15, pAKD18, and pAKD29). We proposed a hypothesis for the evolution of these Tn501-bearing IncP-1β plasmids that predicts recent diversification followed by worldwide spread. Our study increases the available collection of complete IncP-1 plasmid genome sequences by 50% and will aid future studies to enhance our understanding of the evolution and function of this important plasmid family.     

ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.     

A simple procedure for transferring genes cloned in Escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK

A simple method to transfer non-conjugative Escherichia coli plasmids to other Gram-negative bacteria and their maintenance is described. This method involves generation of inverse transposition-mediated cointegrates of the non-conjugative E. coli plasmid with a conjugative IncW broad-host-range plasmid, R388, carrying Tn10. Isolation of such cointegrates was readily effected by conjugal transfer from an E. coli donor containing the two plasmids to an E. coli recipient, with selection for transconjugants expressing a marker of the E. coli plasmid. This method is particularly useful when large series of E. coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination. Utility of the method is shown by a functional analysis in Pseudomonas putida of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. I. Localisation and mutual interactions of four Clo DF13 incompatibility regions

Summary The incompatibility properties of the bacteriocinogenic plasmid Clo DF13 have been examined. By using Clo DF13, Clo DF13 deletion, and transposon insertion mutants as well as compatible R plasmids into which Clo DF13 fragments have been cloned, we could identify and localise four different incompatibility regions on the Clo DF13 genome. These regions, designated incA, incB, incC, and incD are located in the following positions: incA about incD between 1.8% and 9% of the Clo DF13 genome. We studied the contribution of each of the four inc regions, separately and/or in combination with each other, to the incompatibility between two plasmid replicons. Two types of incompatibility can be distinguished: Type I evoked by incD, that overlaps the replication control area of Clo DF13 and type II, caused by incA, B and C. From our observations we present a model for plasmid incompatibility based on a combination of the existing repressor dilution and membrane attachment models.     

Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. A potential role for an 11 base-pair consensus sequence in the excision process

Three novel mitochondrial excision-amplification plasmids of Podospora anserina were identified and the excision-junction sites on the mitochondrial genome determined. All three plasmids were at least partially derived from a common region of the mitochondrial genome termed EcoRI-7 (E7). The entire 5651 base-pair sequence of E7 is presented. Included within this sequence are the E7-specific excision-junction sites of these novel plasmids, the localizations of nine tRNA genes, and the localization of a class I intron of the large rRNA mitochondrial gene. The E7 region contains the 3' portion of this large rRNA gene. Formation of these three novel plasmids as well as other previously described mitochondrial plasmids was found to be associated with the presence of an 11 base-pair consensus sequence, GGCGCAAGCTC, or its complementary sequence. A possible role for this consensus sequence and its complement in plasmid formation and the senescence process of Podospora is discussed. A possible role for the tRNA genes in plasmid formation is considered.     

A method of plasmid classification by integrative incompatibility

A method of plasmid classification by integrative incompatibility has been developed. The characteristics of this system are as follows: (i) The conventional plasmids usually used as standards for incompatibility grouping were integrated into the host chromosome to increase stability and to minimize recombination with the superinfecting plasmid. Strains were constructed by integrative suppression which was in some cases facilitated by the introduction of Tn5 into the plasmid. (ii) The resulting Hfr strains were made deficient in the rec A function to eliminate homologous recombination between the resident and the superinfecting plasmids. A test plasmid is introduced into these rec A Hfr test strains in the stationary phase of growth. In an incompatible cross, the number of transconjugant colonies was usually less than 10^?2 of that in a compatible cross. Occasionally, an inhibitory mechanism, other than incompatibility was coded by the resident plasmid [e.g., restriction in R124 (inc FIV)]. This complicated the interpretation, but did not invalidate the experiment. The colonies arising in incompatible crosses were shown to carry drug resistance determinants coded by both the resident and superinfecting plasmids. These were presumably the result of rec-independent integration of all or part of the superinfecting plasmid into the host chromosome. Thus the reduced frequency of superinfectant formation in an incompatible cross is usually the consequence of incompatibility between the resident and the superinfecting plasmids. This integrative incompatibility system should be useful for epidemiological studies of R plasmids.     

Relationships between autonomous and integrated forms of tetracycline resistance plasmid in Staphylococcus aureus.

Recombinant plasmids carrying apparently the complete genome of a small staphylococcal plasmid, pT181, or of its temperature-sensitive replication mutant, pSA0301, were isolated and characterized; in these recombinants, pT181 or pSA0301 were considered as “integrated” into the other plasmid, inasmuch as they seem to have a subsidiary role in the replication of the respective recombinant plasmids. Using these recombinants, the incompatibility relationships between integrated and autonomous forms of the same plasmid were studied. The results obtained showed that, although integrated plasmids express their incompatibility toward autonomous ones, they are not susceptible to the incompatibility manifested by an autonomous or another integrated plasmid. No differences were observed between pT181 and pSA0301 in their response to the incompatibility manifested by recombinant plasmids. The expression of the incompatibility of an integrated plasmid did not require the function of the repC gene, involved in plasmid autonomous replication. Moreover, the pT181 repC⁺ gene seems not to be expressed when pT181 is integrated into another plasmid in that the integrated form does not complement autonomous pSA0301 for replication at nonpermissive temperature.     

Analysis of the replicon region and identification of an rRNA operon on pBM400 of Bacillus megaterium QM B1551

An 18 633 bp region containing the replicon from the approximately 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non-essential plasmid DNA has implications for systematics and evolutionary biology.