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

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

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430

The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.     

Evolution of the Replication Regions of IncI{alpha} and IncFII Plasmids by Exchanging Their Replication Control Systems

The basic replicons of bacterial plasmids consist of two setsof genetic systems, the replication-structural system and thereplication control system. Comparison of nucleotide sequencessuggested that the basic replicons of plasmids P307 (IncFI)and pMU2200 (IncZ) were generated by reciprocal recombinationbetween ancestors of R100 (IncFII) and ColIb-P9 (IncI), or viceversa. The plasmids of each pair, P307/pMU2200 and R100/ColIb-P9,are structurally unrelated to each other. Based on this information,we constructed in vitro and analyzed P307-like chimeric repliconsfrom ColIb-P9 and R100. When the replication-structural regionof ColIb-P9 was combined with the whole replication controlregion of R100, the resultant replicon replicated stably asR100 did. These results revealed that the basic replicons ofthe plasmids diverged by exchanging their replication controlsystems. Thus, we propose that the replication control systemsof plasmids, in some cases, evolved independently of their structuralsystems, although these two systems work together to maintainthe replication functions. We also showed that the reciprocalrecombination was specified by the unique secondary structuresof RNA involved in the control of expression of the genes encodingthe replication initiator proteins.     

Antirestriction activity of the IncI1 transmissive plasmid R64 ArdA protein

The transmissive plasmid IncI1 R64 contains the ardA gene encoding the ArdA antirestriction protein. The R64 ardA gene locating in the leading region of plasmid R64 has been cloned and their sequence has been determined. Antirestriction proteins belonging to the Ard family are specific inhibitors of type I restriction-modification enzymes. The IncI1 ColIb-P9 and R64 are closely related plasmids, and the latter specifies an ArdA homologue that is predicted to be 97.6% (162 residues from 166) identical at the amino acid sequence level with the ColIb = P9 equivalent. However, the R64 ArdA selectively inhibits the restriction activity of EcoKi enzyme leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The ColIb-P9 ArdA inhibits restriction endonuclease and methyltransferase activities simultaneously. It is hypothesized that the ArdA protein forms two complexes with the type I restriction-modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonuclease. The association of the ColIb-P9 ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of the R64 ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.     

Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.     

Isolation of the replication and partitioning regions of the Salmonella typhimurium virulence plasmid and stabilization of heterologous replicons.

Although the virulence plasmid of Salmonella typhimurium has a copy number of one to two per chromosome, plasmid-free segregants are produced at a rate less than 10(-7) per cell per generation. Three regions appear to be involved in the maintenance of this virulence plasmid. The first two, repB and repC, are functional replicons hybridizing with IncFII and IncFI plasmids, respectively, neither exhibiting the segregational stability of the parent virulence plasmid. The third region, par, cloned as a 3.9-kilobase Sau3A fragment, is not a functional replicon but exhibits incompatibility with the virulence plasmid. Subsequent tests revealed the ability of this 3.9-kilobase par insert to increase the stability of pACYC184 in S. typhimurium from less than 34% to 99% plasmid-containing cells after 50 generations. In addition, the par region increased the stability of oriC, R388, and repC replicons in both S. typhimurium and Escherichia coli hosts. The par region encodes 44,000- and 40,000-molecular-weight proteins essential for the Par+ phenotype but not for the Inc+ phenotype. Although actual sequestering of plasmids within the cell was not demonstrated, all results indicate that the par region described is an actual partitioning locus, similar in organization to those described for plasmids F, P1, and NR1.     

Detection of primase specified by IncB plasmid R864a.

Plasmid R864a (IncB) contains nucleotide sequences homologous with the sog primase determinant of IncI alpha plasmid ColIb-P9. Extracts of Escherichia coli carrying a mutant R864a derepressed for transfer functions showed enhanced primase activity, and contained a large polypeptide identical in size (apparent Mr = 220,000) to the IncI alpha sog gene product.     

Alleviation of type I restriction in the presence of plasmids of group inc I. General characteristics and molecular cloning of the ard gene

The capability of a number of plasmids of incN and incI groups to alleviate an action of type I EcoK, EcoB, EcoD, and EcoA restriction endonucleases on the unmodified DNA was revealed. The efficiency of EcoK action on lambda 0 DNA is alleviated about 10 divided by 100 fold in E. coli K12 AB 1157 bacteria containing the plasmid of incN group (pKM101, N3, pJA4733) or incI group (R144, R648; R621a; ColIb-P9). We have cloned ard gene of ColIb-P9 plasmid (SalI-C fragment) in pBR322 multicopying vector. A hybrid clone abolishing the EcoK restriction has been received. Ard gene activity is independent of the recA, recBc, recF, lexA, umuC, lon bacterial genes activity. Ard gene's product does not inhibit the EcoK restriction endonuclease action as well as ocr protein (phage T7) and does not increase the process of methylation of DNA as well as ral protein of phage lambda.     

Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1

The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific. These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.     

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair

Summary The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5.While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc ^- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.Abbreviations MC mitomycin C - ICS induction of colicin synthesis     

The stb operon balances the requirements for vegetative stability and conjugative transfer of plasmid R388

The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed.     

Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2.

A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner. This region, designated par, consists of two divergently arranged operons: parCBA and parDE. The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division. The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown. It has been proposed that the parCBA operon encodes a plasmid partitioning system (M. Gerlitz, O. Hrabak, and H. Schwabb, J. Bacteriol. 172:6194-6203, 1990; R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac). The intact 3.2-kb region provided the highest degree of stability in the two strains tested. The ability of the parCBA or parDE region alone to promote stable maintenance in the E. coli strains was dependent on the particular strain and the growth temperature. Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon. To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing. In E. coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region. However, in the case of E. coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance. To examine the basis for the apparent differences in postsegregational killing between the two E. coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein. Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E. coli MV10delta lac. A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies. This plasmid was found to be lost from E. coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons.

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a "common" plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with > 80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

Protein-nanocrystal conjugates support a single filament polymerization model in R1 plasmid segregation

To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. We found that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmids segregating as a unit.     

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.     

Generation of miniplasmids from copy number mutants of the R plasmid NR1.

Small, closed circular deoxyribonucleic acid molecules, called miniplasmids, were observed in Escherichia coli harboring copy number mutants of the R plasmid NR1 after growth in medium containing tetracycline. The level of tetracycline resistance conferred by the copy mutant plasmids was lower (3 to 6 microgram/ml) than that conferred by NR1 (100 MICROGRAM/ML). The presence of the miniplasmid enhanced the level of tetracycline resistance conferred by the copy mutant. Miniplasmids of molecular weights 4 X 10(6) to 13 X 10(6) were found. They carried no antibiotic resistance markers and could be eliminated by growth in the presence of chloramphenicol and/or streptomycin-spectinomycin. Studies with the restriction endonucleases EcoRI and Sal I indicated that the miniplasmids are derived from the region of the copy mutant plasmids that contains the origin for replication of the resistance transfer factor. There were approximately 12 copies of the miniplasmid per chromosome, compared with 3 and 6 copies of the copy mutants of NR1. The miniplasmids appeared to be incompatible with the copy mutant plasmids.