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.     

Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1.

Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.     

New incompatibility groups for Staphylococcus aureus plasmids

Incompatibility relationships between naturally occurring staphylococcal plasmids conferring erythromycin or kanamycin resistance have been studied making use of recombinants between these plasmids and pSA0301, a temperature-sensitive mutant plasmid determining tetracycline resistance. The four plasmids encoding kanamycin resistance fall in two incompatibility groups; similarly, the three plasmids responsible for erythromycin resistance belong to two other incompatibility groups. This brings the number of distinct incompatibility groups reported for Staphylococcus aureus plasmids to 13.     

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.     

Characteristics and function of thick and thin conjugative pili determined by transfer-derepressed plasmids of incompatibility groups I1, I2, I5, B, K and Z

Eleven transfer-derepressed plasmids from incompatibility groups I1, I5, B, K and Z were constructed using the dnaG3 mutant Escherichia coli strain BW86. All were found to determine thin flexible and thick rigid pili constitutively. Immune electron microscopy was used to relate thick and thin pilus serotypes with incompatibility grouping. Mutant plasmids that determined only thick pili constitutively transferred efficiently on an agar surface but not in a liquid, whereas plasmids with both kinds of pili transferred equally well in both environments. A mutant of the IncI2 plasmid R721 determined thin pili constitutively, and thick pili at a repressed level, as indicated by electron microscopy. Experiments with this indicated that thin pili were apparently not involved directly in conjugation but were only used to stabilize mating aggregates.     

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.     

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.     

Evolution of bacterial surface exclusion against incompatible plasmids

Many conjugative transferable plasmids exhibit surface exclusion against plasmids of the same incompatibility group. A mathematical model is developed to calculate under which conditions surface exclusion against incompatible plasmids can evolve. It appears that plasmids inducing surface exclusion can evolve and even replace non-excluding plasmids if the copy number is low and the transfer rate high provided that the cost of surface exclusion is small. They can more easily expel the non-excluding plasmids if the possession of a plasmid is not very harmful for a bacterium and if the rate at which plasmids are lost is small.     

Isolation and complementation of temperature-sensitive replication mutants of Staphylococcus aureus plasmid pC194

Temperature-sensitive replication (Tsr) mutants have been isolated from the Staphylococcus aureus plasmid pC194. For three of the four mutant plasmids tested (pSAO801, pSAO802, and pSAO804) the segregation kinetics suggested a complete block of plasmid replication at 43 degrees C. The replication defects of three mutant plasmids: pSAO802, pSAO803, and pSAO804 could be complemented by recombinant plasmids carrying a segment from either the wild type or the other mutant, pSAO801. There was no complementation when the segment carried by the recombinant plasmid was derived from one of the three complementable mutants. These data were taken as evidence for the involvement of a diffusible, plasmid-encoded product, RepH, in pC194 replication. The complementation of the fourth Tsr mutant, pSAO801, could not be tested due to an abnormal susceptibility of this mutant to the incompatibility expressed by recombinants carrying segments derived from pC194 or its mutants. A single mutation was found to be responsible for both pSAO801 instability and its altered incompatibility properties but the nature of the defect has not yet been elucidated.     

Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability

In Agrobacterium tumefaciens, the stability of Ti plasmids differs depending on the strain. So far, little is known about genes that cause the difference in stability. The repABC operon is responsible for replication and incompatibility of Ti plasmids. We constructed recombinant plasmids carrying the repABC operon and different portions of pTi-SAKURA. Cells having the recombinant plasmids that harbored a 2.6-kbp NheI fragment of pTi-SAKURA were found to be transformed via conjugation 100-fold less frequently with a small incompatible repABC plasmid than cells having the recombinant plasmids lacking the 2.6-kbp NheI fragment. Since the phenomenon occurred only when the resident and incoming plasmids belonged to the same incompatibility group, it was suggested that the 2.6-kbp NheI fragment bears the potential enhancing incompatibility. The fragment contained an operon consisting of two open reading frames, tiorf24 and tiorf25. tiorf24 is an orphan gene, whereas tiorf25 is a homologue of a group of plasmid stability genes. Removal of the 2.6-kbp fragment from the resident pTi-SAKURA increased the resident plasmid ejection ratio by the incoming repABC plasmid, whereas addition of the fragment to pTiC58 decreased the ejection ratio, and the loss ratio during growth at 37 degrees C. These data suggest that tiorf24 and tiorf25 are responsible for the stability of pTi-SAKURA, and reduce, in the host bacterium, the frequency of ejection of the resident plasmid, presumably through an incompatibility mechanism.     

The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region.

The incompatibility properties of IncFII R plasmid NR1 were compared with those of two of its copy number mutants, pRR12 and pRR21. pRR12 produced an altered incompatibility product and also had an altered incompatibility target site. The target site appeared to be located within the incompatibility gene, which is located more than 1,200 base pairs from the plasmid origin of replication. The incompatibility properties of pRR21 were indistinguishable from those of NR1. Lambda phages have been constructed which contain the incompatibility region of NR1 or of one of its copy mutants fused to the lacZ gene. In lysogens constructed with these phages, beta-galactosidase was produced under the control of a promoter located within the plasmid incompatibility region. Lysogens containing prophages with the incompatibility regions from pRR12 and pRR21 produced higher levels of beta-galactosidase than did lysogens containing prophages with the incompatibility region from the wild-type NR1. The introduction into these inc-lac lysogens of pBR322 plasmids carrying the incompatibility regions of the wild-type or mutant plasmids resulted in decreased levels of beta-galactosidase production. For a given lysogen, the decrease was greater when the pBR322 derivative expressed a stronger incompatibility toward the plasmid from which the fragment in the prophage was derived. This suggested that the incompatibility product acts on its target to repress gene expression in the plasmid replication region.     

Comparative biology of IncQ and IncQ-like plasmids.

Plasmids belonging to Escherichia coli incompatibility group Q are relatively small (approximately 5 to 15 kb) and able to replicate in a remarkably broad range of bacterial hosts. These include gram-positive bacteria such as Brevibacterium and Mycobacterium and gram-negative bacteria such as Agrobacterium, Desulfovibrio, and cyanobacteria. These plasmids are mobilized by several self-transmissible plasmids into an even more diverse range of organisms including yeasts, plants, and animal cells. IncQ plasmids are thus highly promiscuous. Recently, several IncQ-like plasmids have been isolated from bacteria found in environments as diverse as piggery manure and highly acidic commercial mineral biooxidation plants. These IncQ-like plasmids belong to different incompatibility groups but have similar broad-host-range replicons and mobilization properties to the IncQ plasmids. This review covers the ecology, classification, and evolution of IncQ and IncQ-like plasmids.     

Formation of HfrH-type donor cells as a result of integrative suppression by R-F recombinant plasmids.

Three recombinant plasmids, resulting from recombination between an R plasmid of the FI incompatibility group and the F of HfrH, were introduced in a temperature-sensitive dnaA mutant to isolate Hfr-type-donors. All of the temperature-insensitive clones isolated from two of the three recombinant plasmids had the same origin and transfer pattern as the parental HfrH strain.     

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.     

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.     

Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNA II

Two elements, the products of genes copG and rnall , are involved in the copy-number control of plasmid pLS1. RNA II is synthesized in a dosage-dependent manner. Mutations in both components have been characterized. To determine the regulatory role of the two genes, we have cloned copG , rnall or both elements at various gene dosages into pLS1-compatible plasmids. Assays of incompatibility towards wild-type or mutant pLS1 plasmids showed that: (i) the rnall gene product, rather than the DNA sequence encoding it, is responsible for the incompatibility, and (ii) CopG and RNA II act in trans and are able to correct up fluctuations in pLS1 copy number. A correlation between the gene dosage at which the regulatory elements were supplied and the incompatibility effect on the resident plasmid was observed. The entire copG-rnall circuit has a synergistic effect when compared with any of its components in the correction of pLS1 copy-number fluctuations, indicating that, in the homoplasmid steady-state situation, the control of pLS1 replication is exerted by the co-ordinate action of CopG and RNA II.     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.     

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.     

The distribution of beta-lactamase genes on plasmids found in Pseudomonas.

Seven types of β-lactamases distinguished by analytic isoelectric focusing have been found on 24 Pseudomonas plasmids belonging to at least eight incompatibility groups. TEM-1- and TEM-2-type enzymes that are determined by transposable genetic elements are distributed among five different incompatibility groups. The other β-lactamase types are found on plasmids in single incompatibility groups. β-Lactamases unique to Pseudomonas plasmids occur on plasmids not transmissible to enterobacteria by conjugation.     

Isolation and characterization of a higher-copy-number mutant of plasmid R6K

A stable copy-number mutant (pNH601) of plasmid R6K was isolated by selection for increased resistance to ampicillin determined by this plasmid. The size of the mutant plasmid was found to be unchanged (26 Mg/mol) but it is present in 27 copies of pNH601 perE. coli K-12 chromosome which represents a two-fold increase of R6K copy number value. The following genetic properties of pNH601 are reported and compared with those of R6K: conjugative transfer, fertility inhibition of plasmids belonging to other incompatibility groups, incompatibility with plasmid R485 under both non-selective and selective conditions and the integrative suppression of thednaA ts mutation. The mutant plasmid pNH601 was found to be different from the original R6K in most of these properties.