The mobilization and origin of transfer regions of a Thiobacillus ferrooxidans plasmid: relatedness to plasmids RSF1010 and pSC101

The components for the mobilization function of a plasmid DNA during conjugation include a cis-acting sequence (the origin of transfer, oriT) and a transacting sequence coding for mobilization (Mob) proteins. By genetic and deletion analysis, we have located the mobilization region of pTF1, a cryptic plasmid previously isolated from a Thiobacillus ferrooxidans strain. Within a 2797 bse-pair sequenced region, several open reading frames (ORFs) were predicted; two of the ORFs are divergently transcribed and they encode proteins of calculated molecular masses, 42.6kD (ORF2) and 11.4kD (ORF6). Surprisingly, these protein sequences are substantially similar to two of the previously characterized mobilization proteins of the Escherichia coli IncQ plasmid, RSF1010. Moreover, the pTF1 ORF2 (now designated MobL) sequence is also found to be similar to a presumed mobilization protein of plasmid pSC101. Regions of sequence identity of plasmids pTF1, RSF1010 and pSC101 include their oriT sites. By alkaline agarose gel electrophoresis and DNA sequencing, we have established the location of the relaxation complex nick site within the oriT of pTF1. An identical nick site, which is adjacent to a characteristic 10 base-pair inverted repeat sequence, is also found for plasmid RSF1010. A recombinant plasmid containing a 42 base-pair synthetic piece of DNA encompassing the pTF1 inverted repeat and nick sequence was shown to be oriT-active.     

Control of tryptophan synthetase amplified by varying the numbers of composite plasmids in Escherichia coli cells.

Using pSC101, RSF1010, RSF2124 and RP4 plasmids as vectors and bacteriophage lambdatrpD-A60-3 DNA as a source of the Escherichia coli whole tryptophan operon, composite plasmids of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were constructed in vitro with EcoRI restriction endonuclease and DNA ligase. Each composite plasmid could be maintained stably in E. coli cells. The copy number of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were 4.2, 11.2, 11.9 and 1.6 per chromosome respectively. The tryptophan synthetase activities in cells containing pSC101-trp, RSF1010-trp, RSF2124-trp aand RP4-trp plasmid were found to be 2.1, 6.0, 5.0 and 2.5 times compared with the level in chromosomal trp+ cells when they were grown in a minimal medium. By partial derepression with indolylacrylic acid, the enzyme levels were elevated to 10.1, 16.3, 15.3, 12.3 times, respectively, that of the control cells. The tryptophan synthetase activities did not increase in proportion to the copy number of the plasmids, but were strongly affected by the repression system of host cells.     

The transformation of legionellae by plasmid DNA

For the first time the possibility of the genetic transformation of L. pneumophila and L. bozemanii strains with the use of purified DNA of plasmids pUC19, pUC4K, pSC101 and RSF1010-pBR322 was shown. The frequency of transformation varied from 5.2 x 10(-6) to 5.8 x 10(-7), depending on the strain used in the experiment and plasmid DNA. In some of the transformants obtained in this investigation plasmid DNA whose molecular weight was similar to that of the plasmid DNA used for transformation was detected. The relatively stable preservation of plasmids pSC101 and RSF1010 in Legionella strains and the loss of plasmids pUC19, pUC4K and pBR322 in 80% of transformants during storage were shown.     

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders.

Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally active Agrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

Background  

RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet.     

Genetic analysis of the inter-relationship between plasmid replication and incompatibility

Summary The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050. Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties. Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety. Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1.The inability of the composite plasmid to replicate at 42° in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component. Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell. Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nevertheless retain pSC101 incompatibility. In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Nalidixic Acid

During the conjugal transfer of the R64-11 plasmid at 42 C from donor cells thermosensitive for vegetative deoxyribonucleic acid (DNA) synthesis to recipient minicells, the plasmids are conjugally replicated in the donor cells. This conjugal replication is inhibited by nalidixic acid, and the degree of inhibition is comparable to the reduction in the amount of plasmid DNA transferred to the recipient minicells in the presence of the drug. In addition, the size of DNA transferred to the minicells and the fraction of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA under alkaline conditions are both reduced by nalidixic acid. When the drug is added to a mating that is underway, the rate of conjugal replication is immediately reduced. This change is accompanied by a reduction in the amount of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA. Furthermore, conjugally replicated plasmid DNA that is not associated with the donor cell membrane becomes membrane bound after the addition of nalidixic acid.     

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.     

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.     

Interaction of the IciA protein with AT-rich regions in plasmid replication origins.

A set of AT-rich repeats is a common motif in prokaryotic replication origins. We have screened for proteins binding to the AT-rich repeat region in plasmids F, R1 and pSC101 using an electrophoretic mobility shift assay with PCR-amplified DNA fragments from the origins. The IciA protein, which is known to bind to the AT-rich repeat region in the Escherichia coli origin of chromosome replication, oriC, was found to bind to the corresponding region from plasmids F (oriS) and R1, but not to pSC101. DNase I footprint analysis showed that IciA interacted with the AT-rich region in both F and R1. When the IciA gene was deleted, the copy number of plasmid F increased somewhat, whereas there was no major effect on the replication of pSC101 and R1, or on the E. coli chromosome.     

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.     

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two-component system regulator

We identified a gene ( dpiA destabilizer of plasmid inheritance) which, when overexpressed in Escherichia coli , destabilizes the inheritance of pSC101 and other iteron-containing plasmids as disparate as mini-F and RK6 but not the inheritance of P1, RSF1010 and ColD. These effects of DpiA, which functions like an effector protein for a previously undescribed two-component signal transduction system, were reduced by mutations known to promote pSC101 replication and partitioning. dpiB , a gene encoding the putative histidine kinase of this two-component system, is located immediately 5' to dpiA and adjacent to a DpiA-induced target promoter that transcribes genes having homology to citrate lyase operon genes, citC , citD and citE , of Klebsiella pneumoniae . Disruption of dpiB reversed or reduced the effect of DpiA overproduction on pSC101 inheritance. A second DpiA target, the promoter for a gene ( appY ) implicated in E. coli's response to anaerobiosis, is repressed by DpiA. A mutation in dpiA at a site commonly conserved and phosphorylated in two-component system effector proteins abolished the effects of DpiA overproduction on pSC101 inheritance and negative regulation of appY expression. Our findings suggest a possible mechanism by which environmental and/or cellular stimuli may influence plasmid inheritance.     

Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors

Two cloning vector plasmids, pHSG415 (7100 bp) and a lambda phage cos site-containing derivative (cosmid) thereof, pHSG422 (8760 bp), were constructed from a low copy number plasmid (pSC101) replicon to permit the propagation of cloned DNA segments at low gene dosage levels. Two features of the vectors, namely temperature sensitivity of replication and inability to be mobilized by conjugative plasmids, cause them to exhibit a high level of "biological containment". The essential characteristics of pHSG415 and pHSG422 may be summarized as follows: (1) their genome copy number is low (4--6 copies/chromosome); (2) their replication ceases at high temperature and they are rapidly lost from host cells grown at temperatures of 37 degrees C and above; (3) the relaxation nick site of pSC101, which is thought to be synonymous with its origin of transfer replication, is absent from the vectors; as a consequence, they are not mobilized to a significant extent by co-existing conjugative plasmids that are able to mobilize wild-type pSC101; (4) they contain unique insertion sites for DNA fragments generated by the following restriction endonucleases: EcoRI, XhoI, XmaI, HindIII and PstI; pHSG415 additionally contains single BamHI, BstEII and HincII sites and may also be used to clone PvuI-generated fragments; (5) the plasmids confer upon their host cells resistance to chloramphenicol, kanamycin and ampicillin, and every unique cloning site, except those of BamHI and BstEII, is located within one of these antibiotic-resistance genes.     

Repetitive plasmid sequences generate DNA fingerprinting patterns in mammals

Bacterial plasmids with stringently regulated copy numbers have directly repeated DNA sequences, termed iterons, in the vicinity of their replication origins. These sequences bind a specific protein exerting a key role in the initiation of plasmid replication. Plasmids P1, pSC101 and RFS1010 have different iteron sequences and belong to three different incompatibility groups. Used as DNA probes each of these plasmids generates specific patterns in mammals similar to those obtained by the DNA fingerprinting technique. The iteron-containing regions were identified as the part of the plasmids responsible for those patterns by using polymerase chain reaction (PCR) amplified DNA segments that contained the iteron regions as probes.     

Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid.

A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.     

Genetic organization of the broad-host-range IncP-1 plasmid R751.

We have identified regions encoding conjugal transfer, plasmid maintenance, and trimethoprim resistance on the IncP-1 plasmid R751 by complementation tests with cloned deoxyribonucleic acid fragments and self-replicating derivatives constructed in vitro. The genes for replication and transfer show a scattered organization similar to that previously determined for RK2, another IncP-1 plasmid. Derivatives of RK2 are able to complement R751 derivatives defective in these functions. Restriction enzyme cleavage sites in R751 deoxyribonucleic acid are clustered in regions of the plasmid physical map. Neither region is required for plasmid maintenance or transfer, although one determines resistance to trimethoprim. A similar clustering of cleavage sites is seen with RK2, which nevertheless has a very different restriction map.     

Mutations of temperature sensitivity in R plasmid pSC101.

Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.