Analysis of a region in plasmid R386 containing two functional replicons

A miniplasmid has been obtained from R386 by ligating EcoRI fragments with a fragment carrying a kanamycin-resistance gene. It contains a 6.8-kb Eco fragment of R386 which hybridizes strongly with several IncFI plasmid DNAs but not with the primary or secondary replicons of the F plasmid. This mini-R386 is incompatible with certain IncFI plasmids, and it appears to be one example of a previously unidentified replicon widely distributed in the IncFI group. A region of R386 not closely linked to the 6.8-kb fragment is involved in copy number control of the mini-R386, and a sequence in the same region interacts with mini-F partition functions to cause incompatibility. The 6.8-kb fragment also restricts growth of T7 bacteriophage, and an adjacent fragment restricts phage T4 growth. A further R386 sequence, sharing homology with the F secondary replicon, is capable of autonomous replication. Hence R386, like F, contains at least two functional replicons.     

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.     

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86

Summary Many plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.     

Incompatibility and transforming efficiency of ColE1 and related plasmids

Summary Replicons derived from the ColE1 plasmid are incompatible with one another, but are compatible with their naturally occurring relatives ColK and CloDF13. The incompatibility results in loss, by segregation, of one or the other ColE1 plasmid. In most cases, the smaller derivatives tend to displace the larger ones, and the rate of displacement depends on the difference in size. One mini-plasmid retains only 19% of the sequences of ColE1, yet it exrrts strong incompatibility: other ColE1 plasmids are rapidly lost when it is introduced into the host. The region essential for ColE1 incompatibility is deduced to lie within 700 base pairs of the origin of replication.The transforming efficiency of any ColE1 plasmid is markedly lowered when another incompatible replicon is resident in the competent cells, even when the transforming plasmid is much smaller than the resident.A model of incompatibility is proposed to account for these effects.     

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.     

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.     

Distribution of basic replicons having homology with RepFIA, RepFIB, and RepFIC among IncF group plasmids

Plasmids encoding F-like pili have been divided into groups on the basis of their incompatibility behavior. Three basic replicons have been recognized previously in the IncFI plasmid group and we have now examined their distribution in representative plasmids from 22 of the currently recognized incompatibility groups. The occurrence of these basic replicons was found to be rare outside of the IncF group, and significant hybridization was shown only for RepFIA to IncH1 and I group plasmids. Homology to the RepFIC basic replicon was found in all but one of the IncF group plasmids examined but RepFIA and RepFIB have a more restricted distribution. It appears likely that some plasmids carry vestiges of replicons which still express incompatibility but are incapable of replication. We suggest that evolutionary divergence among the plasmids of the IncF group has resulted from various genetic rearrangements among these basic replicons.     

Molecular homology and incompatibility in the IncFI plasmid Group

The usual grounds for the inclusion of a plasmid in a particular incompatibility group are its mutual incompatibility with a type plasmid of that group, and, in some cases, the demonstration of shared regions of specific homology, presumed to be related to DNA replication. We have found that some plasmids classified as IncFI on genetical grounds share no homology with the previously described incompatibility regions of F on the basis of hybridization of specific radioactive probes to restriction enzyme digests of DNA from these plasmids. Others show homology with some or all of the regions of the F plasmid that can express incompatibility. The incompatibility behaviour of these plasmids has been examined to determine the relationship between the possession of regions of homology and the expression of incompatibility. Three plasmids, ColV3-K30, pHH507 and Entp307, show homology only with the secondary replicon of F and appear to use sequences homologous with the secondary F replicon in their replication. The results are consistent with the propositions that some contemporary IncFI plasmids arose by the integration of several replicons, and, in general, the replicon not being used for replicon expresses its incompatibility, as does the replicon being used for replication. We conclude that incompatibility of two plasmids with F does not necessarily demonstrate relatedness of the plasmids to each other, and that inclusion within the IncFI group can result from the possession of any of several combinations of inc sequences.     

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.     

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids.

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1::Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdA, encoded a polypeptide of 150 amino acids. Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htdA gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.     

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene,and secrete its active gene product

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H. K. Kuramitsu, Plasmid, 1995, 34, 85–95). This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb BglII fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3′-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3′-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.     

The complete nucleotide sequence of the resistance plasmid R478: defining the backbone components of incompatibility group H conjugative plasmids through comparative genomics

Horizontal transfer of resistance determinants amongst bacteria can be achieved by conjugative plasmid DNA elements. We have determined the complete 274,762 bp sequence of the incompatibility group H (IncH) plasmid R478, originally isolated from the Gram negative opportunistic pathogen Serratia marcescens. This self-transferable extrachromosomal genetic element contains 295 predicted genes, of which 144 are highly similar to coding sequences of IncH plasmids R27 and pHCM1. The regions of similarity among these three IncH plasmids principally encode core plasmid determinants (i.e., replication, partitioning and stability, and conjugative transfer) and we conducted a comparative analysis to define the minimal IncHI plasmid backbone determinants. No resistance determinants are included in the backbone and most of the sequences unique to R478 were contained in a large contiguous region between the two transfer regions. These findings indicate that plasmid evolution occurs through gene acquisition/loss predominantly in regions outside of the core determinants. Furthermore, a modular evolution for R478 was signified by the presence of gene neighbors or operons that were highly related to sequences from a wide range of chromosomal, transposon, and plasmid elements. The conjugative transfer regions are most similar to sequences encoded on SXT, Rts1, pCAR1, R391, and pRS241d. The dual partitioning modules encoded on R478 resemble numerous sequences; including pMT1, pCTX-M3, pCP301, P1, P7, and pB171. R478 also codes for resistance to tetracycline (Tn10), chloramphenicol (cat), kanamycin (aphA), mercury (similar to Tn21), silver (similar to pMG101), copper (similar to pRJ1004), arsenic (similar to pYV), and tellurite (two separate regions similar to IncHI2 ter determinants and IncP kla determinants). Other R478-encoded sequences are related to Tn7, IS26, tus, mucAB, and hok, where the latter is surrounded by insLKJ, and could potentially be involved in post-segregation killing. The similarity to a diverse set of bacterial sequences highlights the ability of horizontally transferable DNA elements to acquire and disseminate genetic traits through the bacterial gene pool.     

Nature of the compatible inheritance of hybrid plasmid pAS8 and its deletion mutants with replicon ColE1]

Hybrid plasmid pAS8, that consists of RP4 and ColE1 replicons, is incompatible with RP4 but co-exists with ColE1 replicon. The deletion mutants of pAS8, that replicates under the control of ColE1 replicon only, are incompatible with ColE1 derivatives, although the copy number of pAS8 and its deletion mutants in the cell is the same (1-3 per the chromosome). Incompatibility effect of plasmids is expressed in a sharp decrease in transformant's yield during selection of incoming and resident plasmids markers. In this case incompatibility is a very fast process, that leads to the elimination of resident or incoming plasmid just before plating on the selective medium. On the base of negative control theory, the repressors yield, synthesized in the presence of 1-3 copies of the deletion mutant is enough for the expression of ColE1-specific incompatibility. This ColE1 incompatibility is probably connected with the functional activity of ColE1 replicon. When ColE1 factor replicates under the control of RP4 replicon the expression of ColE1-specific incompatibility does not occur. Possibly, the incompatibility effect takes place when pAS8 deletion mutants cause the synthesis of ColE1-specific repressor. Also, the replicons of ColE1 may competein the membrane attachment site.     

Transfer and stability of drug resistance plasmids inEscherichia coli K12

Mating experiments between pairs of strains ofEscherichia coli containing either the compatible plasmids TP120 (Inc N) and R1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids TP120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and TP113, transfer was only observed in continuous-flow culture systems. Strains ofE. coli containing aggregates of plasmids TP120 and R1 were shown to be stable over 5 months continuous cultivation under carbon limited conditions at a growth rate of 0.1 hours^–1 in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels of resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains ofE. coli initially containing both plasmid TP120 and plasmid R1.     

A replication region of the IncHI plasmid, R27, is highly homologous with the RepFIA replicon of F

A region of the IncHI plasmid R27 has been found to share very close nucleotide sequence homology with the RepFIA replicon of F. This region has been located on a 1.6 kb segment of R27 plasmid DNA, and corresponds to ori-2 and the E gene of F. The incC repeat sequence region shows reduced homology, with the F repeats being an imperfect subset of a larger repeated sequence found in R27. The E gene homologue of R27 is able to initiate replication from the F ori-2 sequence and to repress the E gene promoter of F. The results are consistent with the observed incompatibility behaviour of R27, and have a bearing on the specificity of interaction of E protein with its DNA-binding sites.     

Genetic analysis of transfer and incompatibility functions within the IncHI plasmid R27

This study was undertaken to establish a transfer complementation system for IncH plasmids and to locate regions of incompatibility within the HI1 plasmid, R27. Two regions of R27 were found to contribute to incompatibility as determined by incompatibility testing with fragments of R27 cloned in cosmid vectors. One of these regions hybridized with the IncHI1 rep probe (Couturier et al., Microbiol. Rev. 52, 375-395, 1988). Complementation analysis was carried out using transfer-deficient mutants of R27 in combination with pHH1508a. Cosmid vectors, which contained cloned restriction fragments of R27, were able to complement selected R27 Tra- mutants, enabling the transfer-deficient plasmid to transfer at near-normal frequencies. Complementation of R27 Tra- plasmids by pHH1508a at both 26 and 37 degrees C was shown to occur, but was host-dependent in its degree. These results suggest that the transfer mechanisms of IncHI and IncHII plasmids are related.     

Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids.

Replication of the multicopy mini-R1 plasmid, Rsc11, is dependent on host replication functions dna A, B, C, E and G but independent of polA1. Chloramphenicol immediately stops its replication. A stable relaxation complex is not formed. Composite plasmids were constructed with Rsc11 and other small replicons like pSC101, ColE1 and mini-ColE1. In all combinations the amount of hybrid plasmid DNA in the cell never exceeds the amount of Rsc11 DNA itself. This leads to varying copy numbers of the hybrid plasmids depending on the size of the second plasmid. Replication of the composite plasmids proceeds probably always under the control of the Rsc11 part although the second replicon is still functional. The composite plasmids are incompatible with both the parent replicons.     

Significance of filter mating in integrative incompatibility test for plasmid classification

For classification of plasmids in epidemiological studies, an integrative incompatibility test using liquid mating was developed by Sasakawa et al (Plasmid 3: 116-127, 1980). This test was designed to compare the relative mating frequency of a donor carrying a test plasmid with that of recA recipients carrying various integrated plasmids. To improve the accuracy of this method by increasing transfer frequency of a test plasmid, filter mating was introduced. A transfer frequency 10 to 30,000 times higher than that achieved by liquid mating was attained by filter mating. The degree of increase varied among the incompatibility groups and the majority of members belonging to the same incompatibility group exhibited a similar degree of increase. Standard plasmids were classified correctly with the integrative incompatibility test using filter mating. Out of 26 naturally occurring plasmids of poor transferability in liquid mating, all of domestic animal origin, 25 were correctly classified as IncH with the integrative incompatibility test using filter mating. Moreover, the method is capable of subdividing IncH plasmids directly into IncH1 and IncH2 , because IncH2 , but not IncH1 , plasmids showed incompatibility with the integrated plasmid, R478 , of the IncH2 group.     

Structural and functional features of cis-acting sequences in the basic replicon of plasmid ColIb-P9.

We have structurally and functionally analyzed the cis-elements essential for ColIb-P9 plasmid DNA replication. The putative oriV region encompassed a region of 172 base pairs (bp) located 152 bp downstream of the repZ gene. A typical dnaA box found in this region proved nonessential for the DNA replication of ColIb-P9. The ssi signal of ColIb-P9 is a homologue of the G-sites of R1 and R100 plasmids. Deletion of the G-site led to 1.5-fold reduction of the copy number, suggesting that although this G-site is not essential, it is important for efficient ColIb-P9 DNA replication. In addition, the ColIb-P9 replicon is highly and extensively homologous with the P307 (RepFIC) replicon, and highly homologous with the R100 (RepFIIA) replicon around the G-site region. These facts imply a common ancestry from which the plasmids have evolved.