New replication mutant pnh602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K

A stable deletion derivative pNH602 was obtained when the recently described higher-copy-number point mutant pNH601 of plasmid R6K was introduced to a minicells-producing strain of Escherichia coli. The size of plasmid pNH602 is 18.8 Mg/mol as determined by electron microscopy. The 7.2 Mg/mol fragment of R6K genome missing in pNH602 carries the Smr-determinant and the region finO and, according to the results of restriction analysis, it includes one EcoRI site. With its radioisotopically determined 33 copies of pNH602 per E. coli K-12 chromosome (npc), representing a 23% increase of the point mutant pNH601 and 150% enhancement of R6K npc, plasmid pNH602 differs from another closely related R6K deletion derivative pAS3 of the same size which exhibits only 20 npc. Both pNH602 and pAS3 plasmids are conjugative.     

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.     

Anin vivo cointegrate of two plasmids from incompatibility group X

Formation of a recombinant plasmid designated pNH603 was observed when two plasmids from incompatibility group X, the multicopy plasmid pNH602 (a higher-copy-number deletion derivative of R6K) and the oligocopy plasmid R485, coexisted in a single Escherichia coli cell. According to its size and its restriction endonuclease cleavage pattern, plasmid pNH603 is a true cointegrate of pNH602 and R485. An insertion-sequence-like element coming from plasmid R485 is supposed to mediate the fusion of both replicons. The pNH603 copy number (1-2 per chromosome) indicates that the mechanism of replication of the low-copy-number plasmid is dominant in this cointegrate. No dissociation of pNH603 to parental plasmids was observed even in E. coli K-12 recA+ cells. On the other hand, deletion derivatives of four size classes originate from pNH603 in both recA+ and recA hosts. A miniplasmid designated pNH604, a representative of the most frequent 7 Mg/mol size class, was found, in a low number of copies per host chromosome.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Control of Plasmid R1 Replication: Functions Involved in Replication, Copy Number Control, Incompatibility, and Switch-off of Replication

A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42°C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42°C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 × 10⁶), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.     

Incompatibility properties of the IncFIII/FIV haemolytic plasmid pSU316 when integrated in the Escherichia coli chromosome

The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC::Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA::Tn802) was rapidly lost from strain SU51 both at 30 degrees C and 42 degrees C; (2) the IncFIII plasmid pSU397 (ColB-K98::Tn802) was lost from strain SU51 and at 42 degrees C but not at 30 degrees C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU51. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316.     

Runaway replication of plasmid R1 is not caused by loss of replication inhibitor activity of gene cop.

The replication control functions of a mutant of plasmid R1 that replicates without control at temperatures above 35 degrees C have been analyzed. Although the mutations have not been mapped precisely, the data indicate that the gene (cop) previously identified on the wild-type plasmid (S. Molin and K. Nordström, J. Bacteriol. 141:111-120, 1980) as being responsible for expressing a trans-acting replication inhibitor, as well as for incompatibility of plasmid R1, is not affected in this mutant. Thus, the conditional lack of replication control observed in this plasmid mutant presumably is not caused by the loss of inhibitor activity of the cop gene.     

Genetic heteroduplex analysis of the R6K plasmid]

The results of genetic studies of R6K Tra1- and R6Kdelta[Sm1] mutants of R6K plasmid and those of heteroduplex analysis of DNAs have shown that DNA of this drug-resistant factor contains three loops flanked by the inverted repeats. The latter are designated as IR1, IR2 and IR3 and are of 50, 100 and 120 nucleotides in size respectively. IR1 is inserted into the loop flanked by IR2. Loops with these two repeats are located in major EcoR1 fragment, IR3 having been found in minor EcoRI fragment of the plasmid. The evidence obtained from the analysis of heteroduplex R6K/RSF2124 has shown that the loop with IR1 is corresponding to transposon Tn3. The extent of the deletion deltaSm1 indicates that IR2 may be a part of a transposon bearing the resistance to streptomycin. By comparing present data with those obtaine from the analysis of the RSF1040 factor of DNA replication initiation sites (Grosa et al., 1976), it has been suggested that the loop with IR3 represents a transposon with replicative functions (TnRep). The deletion of the mutant plasmid R6Kdelta[Sm1] (7.2 . 10(6) daltons in size) which affected one of the EcoRI sites not only confers the sensitivity to streptomycin but enhances also the efficiency of conjugational transfer and results in the loss of the R6K ability to bring about integrative suppression and to inhibit the fertility of the plasmids from IncP and IncN groups. The deletion mutant proved to have lost the property of incompatibility with the initial plasmid R6K and with itself.     

Partitioning of the F plasmid: Overproduction of an essential protein for partition inhibits plasmid maintenance

Summary Multicopy plasmids carrying the sopB gene of the F plasmid inhibit stable inheritance of a coexisting mini-F plasmid. This incompatibility, termed IncG, is found to be caused by excess amounts of the SopB protein, which is essential for accuratepartitioning of plasmid DNA molecules into daughter cells. A sopB-carrying multicopy plasmid that shows the IncG⁺ phenotype was mutagenized in vitro and IncG negative mutant plasmids were isolated. Among these amber and missense mutants of sopB, mutants with a low plasmid copy number and a mutant in the Shine-Dalgarno sequence for translation of the SopB protein were obtained. These results demonstrate that the IncG phenotype is caused by the SopB protein, and that the incompatibility is expressed only when the protein is overproduced. This suggests that the protein must be kept at appropriate concentrations to ensure stable maintenance of the plasmid.     

Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids

The promiscuous IncPα plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (O(B)) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995ΔincC. It is a member of the type Ia ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg2? coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 K15A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K15A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995ΔincC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-overexpression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor.     

Copy number control and incompatibility of plasmid R1: Identification of a protein that seems to be involved in both processes

Summary Investigations into the genetic determinants for incompatibility of miniplasmids and hybrid replicons constructed from wild type and mutant R1 revealed the presence of an incompatibility function at the junction of two small PstI fragments. These two fragments were not distinguished in earlier experiments since they have the same mobility on agarose gels. This incompatibility function is distinct from other inc-determinants of R1 (Kollek and Goebel 1979; Molin and Nordström, 1980) and independent of R1-type replication. By means of specific deletions and subcloning of DNA fragments, the location of this new inc-determinant could be determined further. After deletion of this inc-determinant from miniplasmids, a 5-fold increase in copy number was observed which could then be reduced to a copy number of about 1 plasmid per cell by complementation with hybrid plasmids having this function. Incompatibility of miniplasmids deleted in this determinant is not reduced, whereas analogous deletions introduced into recombinant plasmids nearly abolish their incompatibility. This determinant seems to exert strong incompatibility only when cloned on pBR322. Therefore, its main function in plasmid R1 is probably restricted to copy control. The appearance of low copy numbers of miniplasmids carrying this determinant and of trans-acting copy control and strong incompatibility exerted by hybrid plasmids is consistently correlated with the presence of a protein of 11,000 molecular weight, synthesized in relatively large amounts in Escherichia coli minicells.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

Plasmid replication functions

Summary Replicating DNA molecules of the mini R6-5 plasmid, pKTO71, were purified by equilibrium centrifugation in two successive ethidium bromide-caesium chloride gradients, converted to linear forms by cleavage with either HindIII or BglII restriction endonuclease, and examined in the electron microscope. Determination of the replication fork positions in 65 replicating molecules demonstrated that replication is initiated at a unique location on the plasmid and that it proceeds uni-directionally from this site. The direction of replication is such that the origin-proximal BglII cleavage site is replicated late or, in the case of the parent R6-5 plasmid, is such that the R-determinant region of the molecule is replicated early. The origin of replication, located by these experiments at R6-5 coordinate 98.6 kb, is clearly distinct from that of the R6-5 incompatibility determinant which has been shown to be located on an adjacent PstI-generated DNA fragment whose termini have R6-5 coordinates 96.8 and 97.9 kb. This result indicates that the incompatibility function is not an origin DNA sequence.     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Control of plasmid R6K copy numbers in isogenic rep+ and rep Escherichia coli strains

Summary We have analysed as a function of cell doubling times the control of R6K plasmid replication in rep ⁺ and rep strains of Escherichia coli. The rep mutation results in an alteration or loss of an enzyme that unwinds helical DNA. We found in rep ⁺ bacteria that R6K relative dosage (plasmids per genome equivalent) remained nearly constant as growth rates increased. From this we concluded that the average plasmid concentration (plasmids per unit cell mass or volume) fell relative to the average concentration of chromosome origins when growth rates increased. In this context, the control of R6K replication is similar to that of other plasmids as seen by different workers. We also found that the relative dosage of R6K in rep mutants is greater than in rep ⁺ bacteria when both strains were grown at fast growth rates. This finding was expected since at fast growth rates the number of genome equivalents per unit mass is expected to be lower in rep mutants. Unexpectedly, however, we found the effect of the rep mutation on R6K relative dosage had occurred in a step-like manner at a slow growth rate of about 120 min per generation. This implies that both the relative dosage and concentration of R6K had increased in a step-like manner. We also found that the effect of the rep mutation on R6K concentration was lost at fast growth rates while the effect of the mutation on R6K relative dosage was not lost.     

Second replicon in ColV2-K94 mediates the stable coexistence of two incompatible plasmids.

The colicin-producing plasmid pWS12, a Tn903 derivative of ColV2-K94, was found to be incompatible with the IncFI plasmids KLF1 and R386. It was compatible with the IncFII plasmids R538 and R100. Three overlapping mini-ColV derivatives, pWS15, pWS16 and pWS17, were obtained by restriction digestion of pWS12. Unlike pWS12, pWS16 exhibited incompatibility with both IncFI and IncFII plasmids, whereas the pWS15 and pWS17 plasmids expressed IncFII incompatibility but not the IncFI incompatibility of their parental ColV plasmid. We show that, although pWS12 has an IncFII replicon, Rep1, it does not normally express IncFII incompatibility because a second replicon, Rep2 (homologous to the secondary replicon of F), functions during the stable coexistence of the plasmid with IncFII plasmids. When Rep2 is deleted (as in the mini-ColV plasmids) or made nonfunctional (as in a PolA mutant strain), ColV then behaves as an IncFII plasmid.     

Molecular cloning of replication and incompatibility regions from the R-plasmid R6K.

The construction of a physical map of R6K DNA on the basis of specific cleavage of R6K DNA by HindIII and EcoRI restriction endonucleases allowed us to determine the location of the R6K replication and drug resistance regions. Molecular cloning techniques were used to dissect the replication and incompatibility functions of R6K. This R-plasmid possesses two origins of replication, α and β, separated by a stretch of 3900 nucleotides. A region close to ori α. controls the copy number of the composite replicon. Inverted duplications which are 100 to 200 nucleotides long are found at the positions of ori α and ori β, respectively. A 1400-nucleotide long sequence within the region bounded by the inverted duplications and separate from the origins and the control region is involved in the R6K self-replication and replication under conditions of polymerase I deprivation. This region also contains some of the incompatibility genes of R6K. The sequentially asymmetrically bidirectional mode of R6K replication is due to the existence of a replication termination site. This terminator is located outside the sequences bounded by the inverted duplications and is not essential for plasmid DNA replication.     

Plasmids in Nitrobacter

A screening of nine Nitrobacter strains showed that Nitrobacter X14 and Nitrobacter Y possess plasmids designated as pNH1 and pNH2, respectively. The plasmids pNH1 and pNH2 had molecular weights of about 76 Mdal as estimated by agarose gel electrophoresis. They showed similar cleavage patterns when digested with EcoRI, HindIII or BamHI. Electron microscopic investigations exhibited that the plasmid pNH1 had a contour length of 36.9 m corresponding to a molecular weight of 76 Mdal.     

Use of a known plasmid and transposon as tracers for the incompatibility classification of a cryptic plasmid

Summary We have developed a novel genetic technique by which an unknown plasmid can be classified by the use of a plasmid of known incompatibility group. In phenocopy state, cells harboring plasmids of known incompatibility group behave as normal recipients and receive plasmids belonging to the same incompatibility group at a high frequency. This work provides additional evidence that plasmids in phenocopy hosts do not replicate and therefore fail to demonstrate their incompatibility barrier to the incoming plasmids. A cryptic plasmid, now called pWS7, has been identified by this method in a pili, fla female strain of E. coli K12. Genetic analysis shows the plasmid pWS7 is in fact, a sex-factor which is curable with acridine orange. It belongs to the Inc F1 group. Physical analysis confirms its size to be 124 Kb. The plasmid has been labelled genetically with a transposon Tn903 in a recA host and further characterized by heteroduplex analysis. A DNA sequence homology between pWS7 and F'lac plasmid extends only in F-regions, 2.8F-94.5F. The pili, fla host strain of pWS7 shows a high frequency of transformation for recombinant DNA and rapid propagation for a male-specific RNA phage, R17.     

Isolation and characterization of lambda phages carrying the basic replicon of the resistance plasmid R1

Summary Specialized transducing lambda phages, oriR1, harboring DNA from the resistance plasmid R1drd-19 and its copy mutant pKN103 were isolated. From measurements of CCC-DNA content it is concluded that upon infection the phages can establish themselves as self-replicating plasmids in recA hosts lysogenic for lambda. It is thought that this bypassing of lambda immunity is due to the presence of the R1 origin of replication. The plasmids are sensitive to the incompatibility expressed by plasmid R1. This has been shown mainly by transduction of oriR1 into recipients containing R1 plasmids or plasmid pBR322 carrying the basic replicon. We were able to demonstrate that a copy mutant of plasmid R1 was insensitive to copA ⁺, but sensitive to the conserted action of Pst1 fragments F₁ and F₂. This mutant was previously assumed to be of the dominant type. Physical mapping of the oriR1 derivatives verified that they carry the basic replicon of plasmid R1. The plasmids are not stably maintained, but are lost in a frequency of 1%–2% per cell generation, which is consistent with their lack of the R1par region.