Homology of plasmids with a wide range of hosts

According to blotting hybridization and heteroduplex analysis, plasmids R751, R906 and RP4 of Inc Pi group have continuous regions of homology. These homologous regions were mapped on the R751 and RP4-derived pRP401 deletion mutant DNAs. The plasmid pRP401 (m.w. 21.9 kg) retains the broad host range property and has two regions of intensive homology with other Inc P-1 plasmid DNAs. These regions are localized at 8.2-12.0 kb and 13.9-21.9 kb of the physical map of pRP401 plasmid. Homologous regions of pRP401 DNA include at least the replication genes (oriV, trfA, trfB) as well as genes kilB, korA, korB and probably kilC. The data strongly point out that the broad host range plasmids have the same principle of structural and functional organization.     

Comparative analysis of the P-1-group plasmids of incompatibility

Wide host range plasmids (IncP-1) R906, R751 and R702 have several cleavage sites for BamHI, HindIII and EcoRI enzymes, in contrast to RP4 plasmid. Using these enzymes, deletion mutants of R906 plasmid have been obtained in vitro which only lost short DNA fragments (1 to 14 kb). A narrow host range pAV1 plasmid of the same incompatibility group has been transformed into the cells of Escherichia coli. pAV1 is stably maintained in the new host and retains its narrow host range in the course of conjugation. Different restriction fragments of R702, R751, R906 and R906-derived deletion mutants hybridize with the nick-translated probe of RP4 DNA. It is suggested that the wide host range plasmids have a similarity in structural and functional organization.     

Comparative analysis of the structure of incompatibility plasmids N, P and W

Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.     

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties

Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Plasmid pB8 is closely related to the prototype IncP-1beta plasmid R751 but transfers poorly to Escherichia coli and carries a new transposon encoding a small multidrug resistance efflux protein

The IncP-1beta plasmid pB8, which confers resistance to amoxicillin, spectinomycin, streptomycin, and sulfonamides, was previously isolated from a sewage treatment plant. It was found to possess abnormal conjugative transfer properties, i.e., transfer to Escherichia coli by conjugation or electroporation could not be detected. We showed in this study that plasmid pB8 is transferable to E. coli by conjugation, but only at low frequencies and under specific experimental conditions, a phenomenon that is very unusual for IncP-1 plasmids. Determination of the complete 57,198bp pB8 nucleotide sequence revealed that the backbone of the plasmid consists of a complete set of IncP-1beta-specific genes for replication initiation, conjugative plasmid transfer, stable inheritance, and plasmid control with an organisation identical to that of the prototype IncP-1beta plasmid R751. All of the minor differences in the pB8 backbone sequence compared to that of R751 were also found in other IncP-1beta plasmids known to transfer to and replicate in E. coli. Plasmids pB8 and R751 can be distinguished with respect to their accessory genetic elements. First, the pB8 region downstream of the replication initiation gene trfA contains two transposable elements one of which is similar to Tn5501. The latter transposon encodes a putative post-segregational-killing system and the small multidrug resistance (SMR) protein QacF, mediating quaternary ammonium compound resistance. The accessory genes in this region are not responsible for the poor plasmid transfer to E. coli since a pB8 deletion derivative devoid of all genes in that region showed the same conjugative transfer properties as pB8. A Tn5090/Tn402 derivative carrying a class 1 integron is located between the conjugative transfer modules. The Tn5090/Tn402 integration-sites are exactly identical on pB8 and R751 but in contrast to R751 the pB8 element carries the resistance gene cassettes oxa-2 for amoxicillin resistance and aadA4 for streptomycin/spectinomycin resistance, the integron-specific conserved segment consisting of the genes qacEDelta1, sul1, and orf5, and a truncated tni transposition module (tniAB). Although future work will have to determine the molecular basis for the poor transfer of pB8 to E. coli, our findings demonstrate that the host-range of typical IncP-1 plasmids may be less broad than expected.     

Recombination between plasmids of incompatibility groups P-1 and P-2.

R plasmids of incompatibility group P-2 are readily transmissible between Pseudomonas strains, but not to Escherichia coli or other enterobacteria, whereas those of group P-1 have a broad host range. Pseudomonas aeruginosa donor strains carrying both a P-1 plasmid (RP1, RP4, or R751) and a P-2 plasmid (pMG1, pMG2, pMG5, or RPL11) were mated with E. coli K-12, and selection was imposed for resistance markers on the P-2 plasmids. Transconjugants were obtained at a low frequency, in which P-2 markers were expressed and were serially transmissible in E. coli together with P-1 markers. These plasmids had P-1 incompatibility properties, conferred susceptibility to phages active on P-1 carrying strains, and behaved on sucrose gradient centrifugation as unimolecular species of higher molecular weights than the P-1 parent. Recombinant plasmid formation was independent of a functional Rec gene in both donor and recipient and, with R751, had a preferred site leading to loss of trimethoprim resistance. Interaction between insertion sequences may be involved. Thus, plasmids of group P-2 can recombine with R factors of another group quite separate in compatibility properties, host range, and pilus type. Formation of such recombinants provides one pathway by which the genetic diversity of plasmids may have evolved.     

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.     

Genetic and heteroduplex analysis of deletion mutants of plasmid pAS8]

Heteroduplex analysis of deletion mutants of plasmid pAS8 permitted to construct a physical map and to elaborate in greater detail the genetic map of RP4 plasmid. The correlation between the ability of mutants to replicate in cells lacking functional DNA-polymerase I and the length of the deleted segment permitted to map rep genes of RP4 on DNA region with coordinates 9.8-17.3 kb. A relationship between the manifestation of incompatibility of mutants with IncP-1 plasmids and the length of deletions indicates that inc genes(s) are located on DNA region with coordinates 2.1-9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic non-identity of the replication control and incompatibility control in RP4 plasmid. Different degrees of incompatibility manifested by different plasmids suggest a possible polygenic control of the incompatibility.     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

Broad-host-range plasmids from agricultural soils have IncP-1 backbones with diverse accessory genes

Broad-host-range plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to antibiotics and heavy metals or degradation of pollutants. Although some broad-host-range plasmids have been extensively studied, their evolutionary history and genetic diversity remain largely unknown. The goal of this study was to analyze and compare the genomes of 12 broad-host-range plasmids that were previously isolated from Norwegian soils by exogenous plasmid isolation and that encode mercury resistance. Complete nucleotide sequencing followed by phylogenetic analyses based on the relaxase gene traI showed that all the plasmids belong to one of two subgroups (β and ε) of the well-studied incompatibility group IncP-1. A diverse array of accessory genes was found to be involved in resistance to antimicrobials (streptomycin, spectinomycin, and sulfonamides), degradation of herbicides (2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenoxypropionic acid), and a putative new catabolic pathway. Intramolecular transposition of insertion sequences followed by deletion was found to contribute to the diversity of some of these plasmids. The previous observation that the insertion sites of a Tn501-related element are identical in four IncP-1β plasmids (pJP4, pB10, R906, and R772) was further extended to three more IncP-1β plasmids (pAKD15, pAKD18, and pAKD29). We proposed a hypothesis for the evolution of these Tn501-bearing IncP-1β plasmids that predicts recent diversification followed by worldwide spread. Our study increases the available collection of complete IncP-1 plasmid genome sequences by 50% and will aid future studies to enhance our understanding of the evolution and function of this important plasmid family.     

Structure of haloacetate-catabolic IncP-1beta plasmid pUO1 and genetic mobility of its residing haloacetate-catabolic transposon

The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1beta plasmid R751. Comparison of pUO1 with three other IncP-1beta plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.     

DNA recognition by the KorA proteins of IncP-1 plasmids RK2 and R751

The KorA repressor proteins of IncP-1 plasmids belong to a growing family of plasmid-encoded repressors that regulate partitioning genes, and in the IncP-1 plasmids coordinate these with expression of replication and transfer genes as well. Both KorA(RK2) (IncP-1 alpha) and KorA(R751) (IncP-1 beta) recognise the 5'-GTTTAGCTAAAC-3' palindrome. Reporter gene assays showed that KorA proteins from these two main subgroups of IncP-1 plasmids show specificity for their own promoter/operators and this preference was confirmed with in vitro binding studies using gel mobility shift assays on one representative promoter. Class I (high affinity) operators for KorA(RK2) are flanked by an A-A-A/T sequence in the upstream half; the T base was shown to greatly influence strong repression. A C-A-G triplet was present in the same region in the R751 O(A) sequences and the G base was accordingly found to be important for strong KorA(R751) repression. An obvious difference between the two KorA proteins is a histidine to serine change at the C-proximal end of the putative recognition helix of the HTH motif (aa 56). An IncP-1 alpha KorAH56S mutant protein had higher affinity for all operators but had improved more on R751 operators than on RK2 operators. This indicates that KorA of RK2 is not maximised for DNA binding activity and that the aa difference at position 56 may play a role in differentiation between alpha and beta KorA operators.     

Occurrence of deletion plasmids at high rates after conjugative transfer of the plasmids RP4 and RK2 from Escherichia coli to Alcaligenes eutrophus H16

The broad host-range IncP-1 plasmids RP4 and RK2 were transferred by conjugation from Escherichia coli to Alcaligenes eutrophus H16. Among the transconjugants selected on media containing tetracycline, a considerable number did not express kanamycin resistance. By comparing restriction patterns of plasmids isolated from a large number of transconjugants a variety of different deletion derivatives were found. All of these possess more or less extended deletions always including parts of the tra 1-region. The plasmids RP4 and RK2, once established in A. eutrophus H16 showed a high stability and it can be concluded that deletion formation is connected with the conjugation process. Evidence is given that degradation of DNA entering an A. eutrophus recipient cell during the conjugative transfer process may be involved in deletion formation. Furthermore, the finding of a small deletion derivative of RP4 lacking the transacting replication function trfB and the entire kil-kor-system may allow the assumption that these gene functions are not essential for replication and maintenance of RP4 in A. eutrophus hosts.     

The multiple antibiotic resistance IncP-1 plasmid pKJK5 isolated from a soil environment is phylogenetically divergent from members of the previously established alpha, beta and delta sub-groups

The 54,383bp plasmid pKJK5 was recovered from a soil environment by exogenous plasmid isolation and conveys resistance towards tetracycline and trimethoprim. Sequencing and annotation revealed a high level of structural similarity of the backbone genes to other IncP-1 plasmids containing a Tra1 and Tra2 region, a central control module and a replication initiation module. A considerable degree of divergence was associated with the backbone genes of pKJK5 as compared to homologous genes in the alpha, beta and delta subgroups, which indicates that pKJK5 may belong to a novel subgroup of IncP-1 plasmids, which may also accommodate the partially sequenced non-subgroup classified plasmid pEMT3. Individual backbone genes in pKJK5 have a GC-content, which is consistently lower (average 6.3%) than the homologous genes from the archetype IncP-1beta plasmid R751 indicating homogenous amelioration of IncP-1 plasmid backbone genes. Two discrete accessory elements of 2145bp (load 1) and 11678bp (load 2) respectively are situated between the Tra1 and Tra2 regions of pKJK5, both bounded by inverted repeats and direct flanking repeats indicative of transposon-mediated insertion. Load 1 consists of an insertion sequence ISPa17 and load 2 is a Tn402-derivative containing a class 1 integron, IS1326 and a fragment identical to a region of plasmid pTB11 harboring a tetracycline resistance determinant and part of an IncP-1alphaoriV region.     

Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncP beta sub-group are closely related

Summary Comparison of physical maps of the broad host range plasmids R751, R906 and R772, belonging to the IncP sub-group of the Escherichia coli incompatibility group P, reveals two large regions of similarity, separated by dissimilar regions which contain the majority of the cleavage sites for restriction endonucleases with hexanucleotide recognition sites. Mapping of the regions of these plasmids which show homology to probes specific for genetically characterised segments of the distantly related IncP plasmid RK2, involved in plasmid maintenance or conjugal transfer, reveals that all four plasmids share a similar genetic organisation. In each case the homologous plasmid back-bone is interrupted by heterologous segments both between the essential replication loci oriV and trfA, and between the conjugal transfer regions tra1 and tra2, although in the case of R772 the segment of the backbone carrying the trfA and tra2 regions is inverted relative to that of the other plasmids. However, in the case of pJP4, shown to be a fourth member of the IncP sub-group, the back-bone is interrupted only by a single large segment adjacent to the trfA region. Mapping of the regions of the four IncP plasmids which show homology to Tn501 and nucleotide sequence determination at the ends of the homologous regions reveals that R906, R772 and pJP4 share a common mercury resistance region. This region, which appears to have been inactivated in R772, was probably inserted into a common ancestor of these plasmids by the transposition of an element related to an ancestor of Tn501. R751 shows no trace of the mercury resistance region, but contains a short relict of Tn501, derived from an independent insertion event.     

Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways

Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.     

Plasmid transduction using the D3112DeltaH miniphage in Pseudomonas aeruginosa cells]

Plasmid DNA transduction with mini-D3112 delta H, deletion derivative of phage D3112, which lost the genes essential for phage growth but retained the sites required for transposition and packaging was studied. Unlike D3112, mini-D3112 delta H element can transduce plasmids and plasmid markers at frequencies of 10(-5)-10(-8) in rec+ cells of Pseudomonas aeruginosa. Plasmids R1162 and R388 of the size smaller than phage genome were transduced intact. Large plasmids, like RP4 and R151, were deleted under transduction. By this way, we isolated deletion derivatives of RP4. The smallest derivative pN2 contained a 4.5 kb fragment of RP4. Unlike the latter, pN2 plasmid had narrow host range and did not maintain in Escherichia coli cells.     

Development of a system vector-host in methylotrophic bacteria

Cloning of methylotropic and other Gram negative bacteria's genes was performed using vectors derived from IncP4 plasmids. Plasmids, such s RSF1010 are 8.8 kb in length, have a high copy number and broad host range and can be mobilized efficiently by a number of conjugative plasmids. IncP4 plasmids have relatively few restriction enzyme's targets suitable for cloning. In this paper the construction of versatile and special purpose IncP4 vectors available for cloning DNA into broad range of bacterial species are described. The seria of versatile vectors involves the transposon containing plasmid and two-replicon vectors.In genetic construction of special vector for direct cloning of restriction fragments the genetic regulation elements of Tn 1 were used. On the base of IncP4 replicon special vectors for construction of bank genes (cosmids) and the vectors for cloning of regulation sequence were also constructed.