Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.     

In vivo cloning of the pectate lyase and cellulase genes of Erwinia chrysanthemi

Using an RP4 plasmid which carries a mini-Mu prophage which allows it to integrate spontaneously random pieces of its host chromosome, we cloned in vivo at least some of the pectate lyase and cellulase genes of the Erwinia chrysanthemi strain B374. The RP4-prime plasmids were used to localize the cloned genes on the B374 chromosome by co-transposition mapping and to subclone most of the genes in a classic high copy number plasmid vector.     

Replication defective RP4 plasmids recovered after chromosomal integration

pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication. Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen. This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA. It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon. The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations. The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division.     

Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage lambda carrying IS1

In vitro recombination was used to generate RP4 plasmids with an inserted restriction fragment of bateriophage λ. In some cases the λ DNA also carried the insertion sequence IS1. Comparisons were made between the abilities of these plasmids to mobilize the Escherichia coli K-12 chromosome in different genetic backgrounds. RP4-borne IS1 acting alone promoted chromosome transfer but with an efficiency 1% of that resulting from more extensive plasmid-chromosome homology. A recA mutation in the donor depressed the mobilization frequency below the level of detection. Correlation of the direction of chromosome transfer and the orientation of the cloned λ DNA allowed the direction of RP4 transfer to be determined. Studies on recombinants showed that in general they also acquired an intact, autonomous plasmid, suggesting the process of mobilization by RP4 may differ in certain features from chromosome transfer by F.     

Rp4 Promotion of Transfer of a Large Agrobacterium Plasmid Which Confers Virulence

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.     

Chromosomal transfer promoted by the promiscuous plasmid RP4.

We have studied the properties of the recombinant plasmid RP4λatt. This plasmid possesses the EcoRI-generated fragment of phage λ containing the genes att-int-xis (srIλ2–3) inserted into the single EcoRI site of the promiscuous plasmid RP4. The insertion of this λ fragment has no detectable effect on normal plasmid functions. However, it confers the ability to promote low-frequency polarized chromosomal transfer by int-promoted integration into the host λ attachment site attλ. We have succeeded in isolating an Hfr derivative which has the plasmid stably integrated at attλ. The Hfr derivative is unusual in having both an integrated and an autonomous RP4λatt stably coexisting in the same cell.     

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.     

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.     

Map of plasmid RP4 derived by insertion of transposon C.

We have determined the location of 36 sites on plasmid RP4 into which transposon C (an 8.5 × 10⁶ molecular weight DNA sequence conferring trimethoprim and streptomycin resistance) had spontaneously inserted itself. These were located by sucrose gradient analysis of EcoRI-generated and then, separately, the HindIII-generated DNA fragments from each RP4-TnC² plasmid. RP4 has a single EcoRI-susceptible site and, suitably displaced from this, a HindIII-susceptible site, whereas TnC has, respectively, one and two sites for these two enzymes. Thus the sizes of the restriction fragments depend on the location and orientation of the inserted TnC.Some of the RP4-TnC clones had lost one of the RP4 characters: transferability (Tra), tetracycline (Tc) or kanamycin (Km) resistances, but no ampicillin (Ap) sensitive clones were detected. Insertions giving each of these phenotypic changes cluster together at positions on the circular RP4 map that presumably locate the genes responsible for the Tra⁺, Km^r and Tc^r phenotypes. The Tra^? plasmids were grouped into four classes on the basis of their conferred phage sensitivities and plasmid copy numbers. The gene giving Ap^r was located by its known proximity to a BamHI-susceptible site. All the plasmids analysed had TnC inserted with one particular orientation. TnC insertions giving no detectable phenotypic change were not randomly placed around RP4, but clustered into certain regions. Two large regions, one containing TnA, had no TnC insertions. Ligation experiments with restriction fragments from various RP4-TnC plasmids led to the conclusion that both these regions contain genes essential to the replication and maintenance of RP4. The location of the HindIII site of RP4 within the gene giving Km^r should prove valuable to the use of this plasmid as a cloning vehicle.     

Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.     

Plasmid and transposon transfer to Thiobacillus ferrooxidans.

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.     

Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage λ carrying IS1

In vitro recombination was used to generate RP4 plasmids with an inserted restriction fragment of bateriophage λ. In some cases the λ DNA also carried the insertion sequence IS1. Comparisons were made between the abilities of these plasmids to mobilize the Escherichia coli K-12 chromosome in different genetic backgrounds. RP4-borne IS1 acting alone promoted chromosome transfer but with an efficiency 1% of that resulting from more extensive plasmid-chromosome homology. A recA mutation in the donor depressed the mobilization frequency below the level of detection. Correlation of the direction of chromosome transfer and the orientation of the cloned λ DNA allowed the direction of RP4 transfer to be determined. Studies on recombinants showed that in general they also acquired an intact, autonomous plasmid, suggesting the process of mobilization by RP4 may differ in certain features from chromosome transfer by F.     

Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 μM CdCl₂), permitting long-term community monitoring. The broad-host-range IncPα plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd^r or Ni^r density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni^r transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.     

Boundaries of the Origin of Replication: Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replication (ori); pET-28a(+) contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+) replicon–comprising the ori, RNAI, RNAII, and Rom–with the p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. This essential need is currently not filled.     

Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid.     

Ammonia Inhibition of Plasmid pRmeGR4a Conjugal Transfer between Rhizobium meliloti Strains

We have examined nutritional factors influencing conjugal transfer of the two nonsymbiotic large plasmids, pRmeGR4a and pRmeGR4b, of Rhizobium meliloti GR4. To monitor transfer, each plasmid was tagged with a different antibiotic resistance marker. Transfer of plasmid pRmeGR4b was dependent upon the presence of plasmid pRmeGR4a on the same donor cell. Transconjugants for pRmeGR4b were obtained at frequencies 5-to 10-fold higher than transconjugants carrying both plasmids, indicating that mobilization of pRmeGR4b by pRmeGR4a probably occurred in trans. Conjugal transfer of the tagged plasmids between R. meliloti strains was tested on minimal medium supplemented with single amino acids, nitrate, or ammonium as the single nitrogen source. A higher number of transconjugants was obtained when glutamate was the only nitrogen source, whereas conjugation was virtually undetectable on ammonium. No relationship was found between donor or recipient growth rate and plasmid transfer rate on a given nitrogen source. Furthermore, in media containing both glutamate and ammonium as nitrogen sources, transfer was reduced almost 100-fold compared with that in media containing glutamate alone. Inhibition was readily detected at 2.5 mM or higher concentrations of either ammonium chloride or ammonium sulfate and appeared to be specific for exogenously supplied ammonium. Inhibition of conjugal transfer between R. meliloti strains by ammonium was only observed for rhizobial plasmids, not for a heterologous plasmid such as RP4. Apparently, ammonium did not affect the plasmid-encoded transfer machinery, as it had no influence on rhizobial plasmid transfer from R. meliloti to Agrobacterium tumefaciens. The effect of ammonium seemed to take place on R. meliloti recipient cells, thereby reducing the efficiency of plasmid conjugation, probably by affecting mating pair formation or stabilization.     

Construction of conjugative gene transfer system between E. coli and moderately thermophilic, extremely acidophilic Acidithiobacillus caldus MTH-04

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-host-range IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-host-range IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The Km(r) and Sm(r) selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

The use of transposons to introduce well-defined deletions in plasmids: Possibilities for in vivo cloning

A method for obtaining well-defined deletions in an octopine Ti plasmid was developed. It was based on the assumption that deletions would occur between two directly repeated transposons, when both are temporarily present in one plasmid molecule. To obtain such a situation, recombination has been forced between Agrobacterium tumefaciens Ti plasmids, each carrying the transposon Tn1831 at a different position. In a number of cases, most probably when the transposons are directly repeated, deletion formation indeed occurred and at high frequency. Mutants were isolated carrying Ti plasmids with one copy of Tn1831, and the region of DNA, between the positions of the transposons in the original plasmid, deleted. Moreover, in the case that the segment of DNA, enclosed by the two transposons, harbors the requirements for autonomous replication of an R plasmid, it is shown that in vivo cloning of such a segment of the Ti plasmid on the R plasmid can be accomplished.     

F factor inhibition of conjugal transfer of broad-host-range plasmid RP4: requirement for the protein product of pif operon regulatory gene pifC.

By the use of deletions, point mutations, and gene fusions, we show that the protein product of the F factor pifC gene is responsible for F factor inhibition of plasmid RP4 conjugal transfer. Deletion analysis of pif sequences carried by pSC101-F chimeric plasmids demonstrated that removal of all or part of the pifC coding sequence greatly decreased or abolished the ability of these plasmids to inhibit RP4 transfer. Amber mutations in the pifC gene eliminated inhibition in an Su- host strain but not in and Su+ (supF) host. Plasmids carrying nonpolar pifC mutations did not decrease the efficiency of RP4 transfer when present in trans. Whereas pifC+ plasmids inhibited RP4 transfer, the presence of RP4 in the same cell as F' lac increased F'lac Pif activity approximately 1,000-fold. This effect most likely resulted from the binding of the pifC product to RP4 DNA and concomitant derepression of the F factor pif operon. PifC inhibited trans mobilization of pMS204, a nonconjugative plasmid carrying the RP4 oriT locus, by the RP1 derivative pUB307. pMS204 had no trans effect on pif operon expression, whereas pUB307 increased F'lac Pif expression, as did RP4. Our results suggest that the pifC product inhibits expression of one or more RP4 genes, the products of which are required for conjugal transfer of RP4 and are required in trans for mobilization of nonconjugal RP4 oriT containing plasmids.