Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli.

The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein.     

Reciprocal intrapool variation in plasmid copy numbers: a characteristic of segregational incompatibility

An experimental analysis of the concept that incompatible plasmids occupy a common intracellular pool from which copies are drawn at random for replication and assortment is presented. Intrapool variations in an incompatible heteroplasmid strain are inevitable and it is shown that these variations can be exploited by differential selection to amplify one plasmid at the expense of the other. Constant overall copy number is demonstrated for isogenic wild-type replicons and also for isogenic copy mutants whose copy numbers are so great that segregational incompatibility cannot be measured. In the test system used, that of the Staphylococcus aureus plasmid pT181, the rate of replication is probably determined by the availability of a trans-active initiator protein, RepC. In heteroplasmid strains containing wild-type and dominant copy mutant plasmids, although intrapool variation occurs, the total copy number is not constant but varies as a consequence of selection for or against the mutant plasmid. This is because all of the RepC is synthesized from the mutant plasmid (the wild-type is hyper-repressed) and therefore the selection affects the supply of RepC at the same time that it affects the copy number of the plasmid. None of these effects are seen with single plasmids or with compatible pairs.     

Staphylococcus aureus chromosomal mutation specifically affecting the copy number of Inc3 plasmids

A chromosomal mutation leading to an important increase in the copy number of plasmid pT181 and its derivatives has been isolated from Staphylococcus aureus strain 8325. The amplification effect in the mutant strain SA1350 was found to be specific for plasmids of the Inc3 group, to which belongs pT181. There are some other differences in the behavior of Inc3 plasmids between SA1350 and 8325, including stable maintenance in SA1350 at high copy number of temperature-sensitive replication mutants at restrictive temperatures, and altered incompatibility properties. Derivatives of SA1350 carrying only Inc3 plasmid mutants with high copy numbers (Cop mutants) could not be obtained, suggesting a lethal runaway plasmid replication in this situation. SA1350 expressed also a temperature-sensitive phenotype. The relationship of this character to the plaC1 mutation determining the amplification of Inc3 plasmids has not yet been elucidated.     

Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.     

Translational control by antisense RNA in control of plasmid replication

Control of replication of plasmids involves two processes: measurement of the copy number of the plasmid and adjustment of the replication frequency accordingly. For both these processes IncFII plasmids use an antisense RNA (CopA RNA) that forms a duplex with the upstream region (CopT) of the mRNA of the rate-limiting RepA protein. The kinetics of duplex formation was measured in vitro for the wild type and for a cop mutant plasmid; the mutant showed a reduction in the second-order rate constant for the formation of the RNA duplex and a similar increase in copy number. Hence, the kinetics of duplex formation and the concentration of CopA RNA determines the copy number of the plasmid.     

Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control

DNA from the replication control region of plasmid NR1 or of the Inc- copy mutant pRR12 was cloned into a pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia coli cells that harbored either NR1 or pRR12. After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further. By this process, 134 plasmids with Inc- mutations in the cloned NR1 or pRR12 DNA were obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertions in the NR1 DNA and were not examined further. Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NR1-derived replicon (Rep+ or Rep-), (ii) the copy numbers of the Rep+ reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.     

Isolation and characterization of pLS 1 plasmid mutants with increased copy numbers

Abstract Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.     

Control of F plasmid replication by a host gene: evidence for interaction of the mafA gene product of Escherichia coli with the mini-F incC region.

Replication of F (including mini-F) and some related plasmids is known to be specifically inhibited in mafA mutants of Escherichia coli K-12. We have now isolated and characterized mini-F mutants that can overcome the replication inhibition. Such plasmids, designated pom (permissive on maf), were obtained spontaneously or after mutagenesis with hydroxylamine or by transposon (Tn3) insertion. In addition to their ability to replicate in mafA mutant bacteria, the pom mutant plasmids exhibit an increased copy number and resistance to "curing" by acridine dye in the mafA+ host. In agreement with these results, Tn3-induced pom mutants were found to carry Tn3 inserted at the incC region of mini-F DNA, known to be involved in incompatibility, control of copy number, and sensitivity to acridine dye. Furthermore, three of the seven mini-F plasmids tested that carry Tn3 within the tandem repeat sequences of the incC region (previously isolated by other workers) exhibit all the phenotypes of pom plasmids, the ability to replicate in the mafA strain, and high copy number and acridine resistance in the mafA+ strain. The rest of the plasmids that contain Tn3 just outside the tandem repeats remain wild type in all these properties. These results strongly suggest that the putative mafA gene product of host bacteria controls mini-F replication through interaction with the incC region.     

Directed evolution of copy number of a broad host range plasmid for metabolic engineering

Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host.     

Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers.

Mutated forms of trfA, the replication protein gene of plasmid RK2, that support a minimal RK2 origin plasmid in Escherichia coli at copy numbers up to 23-fold higher than normal have been isolated. Six such high-copy-number (copy-up) mutations were mapped and sequenced. In each case, a single base transition led to an amino acid substitution in the TrfA protein primary sequence. The six mutations affected different residues of the protein and were located within a 69-base-pair region encoding 24 amino acids. Dominance tests showed that each of the mutants can be suppressed by wild-type trfA in trans, but suppression is highly dependent on the amount of wild-type protein produced. Excess mutant TrfA protein provided in trans significantly increased the copy number of RK2 and other self-replicating derivatives of RK2 that contain a wild-type trfA gene. These observations suggest that the mutations affect a regulatory activity of the TrfA replication protein that is a key factor in the control of initiation of RK2 replication.     

DnaA protein is not essential for replication of IncFII plasmid NR1.

By transformation of dnaA null mutant host cells that are suppressed either by an rnh mutation or by chromosomal integration of a mini-R1 plasmid, it was shown that replication of miniplasmids composed of the NR1 minimal replicon had no absolute dependence upon DnaA protein. In addition, the suppression of the dnaA null mutation by the integrated mini-R1, which is an IncFII relative of NR1, was found to be sensitive to the expression of IncFII-specific plasmid incompatibility. This suggests that the integrative suppression by mini-R1 is under the control of the normal IncFII plasmid replication circuitry. Although NR1 replication had no absolute requirement for DnaA, the copy numbers of NR1-derived miniplasmids were lower in dnaA null mutants, and the plasmids exhibited a much reduced stability of inheritance during subculture in the absence of selection. This suggests that DnaA protein may participate in IncFII plasmid replication in some auxiliary way, such as by increasing the efficiency of formation of an open initiation complex at the plasmid replication origin. Such an auxiliary role for DnaA in IncFII replication would be different from that for replication of most other plasmids examined, for which DnaA has been found to be either essential or unimportant.     

Plasmid repopulation kinetics in Staphylococcus aureus

We have analyzed the kinetic route by which the indirectly controlled Staphylococcus aureus plasmid, pT181, responds to and corrects fluctuations in copy number. The kinetics of copy number correction from low to steady-state levels (termed repopulation) were determined using two different methods of copy number reduction. Thermosensitive replication (Tsr) mutants of pT181 were grown at nonpermissive temperatures to lower copy number and then shifted to a permissive temperature to allow repopulation. After the downshift, both wild-type and copy mutant plasmids, with active inhibitors, exhibited a burst of exponential replication that resulted in a two- to threefold overshoot of normal steady-state copy numbers. This was followed by inhibition of replication and eventual reestablishment of the steady-state replication rate. Similar replication kinetics were observed when these plasmids were introduced into naive cells by high-frequency transduction. By contrast, a pT181 copy mutant with a nonfunctional inhibitor-target regulation did not overshoot its steady-state copy number, but instead repopulated asymptotically. These results suggest that at low copy numbers, pT181 and its derivatives replicate at near-maximal rates and overshoot prior to the establishment of an inhibitory concentration of repressor. The maximal replication rate is independent of the plasmid's cop genotype. As the copy number increases, inhibitor accumulates and eventually reduces the replication rate. In the absence of an active inhibitor, the steady-state copy number is established at a level that must be limited by some other invariant factor.     

Replication mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B resistance plasmid pT48

Copy-number mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B (MLS) resistance plasmid pT48 were isolated by their resistance to the non-inducing macrolide, tylosin. One mutant plasmid, pcopD3, showed a three- to five-fold cis-dominant increase in copy number, and nucleotide sequence analysis revealed that the mutant had a single base change within the replication region. All other pT48 mutants examined had the unusual phenotype of increased plasmid multimerization and elevated copy number. These mutants were effective in trans and DNA sequencing showed that plasmids with this phenotype were deleted in one of two ways. The deletions caused similar alterations to the C-terminus of the wild-type pT48 Rep protein. The two types of mutant Rep proteins terminate with the same pentapeptide sequence: Ala-Asn-Glu-Ile-Asp. The multimerization phenotype of these mutants can be explained by defective termination of rolling-circle type replication.     

Studies on plasmid replication. III. Isolation and characterization of replication-defective mutants

Some 85 Staphylococcus aureus mutants phenotypically thermosensitive for penicillinase plasmid segregation (Seg-) have been isolated and characterized. Some of the mutations were plasmid-linked and those studied in detail were found to be defective in plasmid replication, most problbly at the initiation stage. Analysis of the segregation behavior of these mutants suggested a figure of 2.7 for the average number of plasmid copies per cell in a random culture. Other mutations were host chromosome-linked and these could be divided into at least three classes on the basis of their ability to maintain plasmids of the two different incompatibility sets: some were defective for type I plasmids, some for type II, and some for both types. One host mutant, defective in segregation of type I but not type II plasmids, was defective in polymerization of both.     

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.     

Evolved plasmid‐host interactions reduce plasmid interference cost

Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad‐host‐range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host's DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts' essential proteins.     

Binding of RepE initiator protein to mini-F DNA origin (ori2). Enhancing effects of repE mutations and DnaJ heat shock protein.

Replication of mini-F plasmid in Escherichia coli requires the plasmid-encoded RepE initiator protein and a number of host factors and is regulated by interaction of RepE with specific sequences near the replication origin, ori2. We have examined DNA binding properties of several hyperactive mutant RepE proteins with single amino acid substitutions. Plasmids carrying these (repE) mutations, unlike the parental plasmid, can replicate in bacterial hosts lacking the heat shock sigma factor (sigma 32) or deficient in the DnaK, DnaJ, or GrpE heat shock protein. Using gel-retardation assays, the mutant RepE proteins were shown to bind the ori2 repeated sequences with much increased affinities compared to the wild type RepE, whereas they bound to the repE operator with slightly reduced affinities. These results agreed well with the properties of mutant RepE proteins studied in vivo and accounted for the high RepE initiator activities and the high copy numbers of mutant plasmids. In addition, the DnaJ heat shock protein was found to markedly enhance the binding of wild type RepE to ori2 or the operator. DnaK protein with or without ATP failed to show such enhancements. Thus, among the heat shock proteins required for mini-F replication, DnaJ appears to play a major role in RepE binding to ori2 and the operator, perhaps accompanied by RepE activation.     

Effect ofEscherichia coliIHF Mutations on Plasmid p15A Copy Number

Four isogenic strains (himAhimDdouble mutant,himAandhimDsingle mutants, and their wild type counterpart) harboringorip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation. The copy number oforip15A plasmid increases about four times in thehimAhimDdouble (65–70 copies per cell) andhimDsingle mutant cells (50–56 copies per cell) and was almost the same inhimAmutant (17–18 copies per cell) and wild type cells (14–16 copies per cell). The results suggest that HimD can form homodimers, which are functionally competent for the regulation oforip15A plasmid copy number. Complementation experiments ofhimAhimDdouble mutant cells using plasmid carryinghimAandhimDgenes (pP_LhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number oforip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant). The absence of IHF (usinghimAhimDdouble mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid.     

Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis.

We have prepared and analyzed two types of gene fusion between the replication initiator gene, repC, and the reporter gene, blaZ, in order to investigate the relationship between pT181 plasmid copy number and RepC initiator protein production. A series of pT181 copy mutant plasmids, with copy numbers ranging from 70 to 800 copies per cell, were analyzed. In one type of gene fusion used in this study, blaZ was translationally coupled to the C-terminal end of the repC coding sequence such that native forms of both proteins were produced. This gene fusion arrangement, which permitted monitoring of RepC production (as BlaZ activity) by plasmids using the protein for their own replication, demonstrated a linear relationship, with one exception, between RepC production and plasmid copy number over a 20-fold range. In the second type of fusion, blaZ was translationally fused to the C-terminal end of repC. As the translational fusion did not produce active RepC protein, the fusion-containing pT181 derivatives were maintained in a strain which provided RepC in trans, and were thus analyzed at constant copy number. In contrast to previous analyses of this type, our translational fusion constructs expressed repC at levels proportional to the copy numbers of the plasmids from which the fusions were prepared. Using these data, we have calculated a minimum figure for the number of RepC molecules synthesized per replication event.