Replication and conjugative mobilization of broad host-range IncQ plasmids

The IncQ plasmids have a broader host-range than any other known replicating element in bacteria. Studies on the replication and conjugative mobilization of these plasmids, which have mostly been focused on the nearly identical RSF1010 and R1162, are summarized with a view to understanding how this broad host-range is achieved. Several significant features of IncQ plasmids emerge from these studies: (1) initiation of replication, involving DnaA-independent activation of the origin and a dedicated primase, is strictly host-independent. (2) The plasmids can be conjugatively mobilized by a variety of different type IV transporters, including those engaged in the secretion of proteins involved in pathogenesis. (3) Stability is insured by a combination of high copy-number and modulated gene expression to reduce metabolic load.     

A molecular analysis of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans

Abstract: A 12.4-kb plasmid, pTF-FC2, that was isolated from Thiobacillus ferrooxidans and which is capable of replication in a wide range of Gram-negative bacteria, has been sequenced. The extent of the regions involved in both replication and mobilization have been delineated. The site of initiation of replication ( oriV ) has been localized on a 185-bp fragment and the origin of transfer ( oriT ) on a 138-bp fragment. Three proteins that were essential for replication and four that were essential for mobilization have been identified. The origin of replication was clearly similar to that of the IncQ plasmids although no complementation or incompatibility between pTF-FC2 and the IncQ plasmid, R300B, was detected. There was a clear similarity in the size,location and amino acid sequence of the proteins of the pTF-FC2 mobilization region with those of the TraI region of the IncP plasmids, RP4 and R751.Two inverted repeated sequences which had 37/38-bp and 38/38-bp sequence identity with the Tn 21 transposon were identified. The C-terminal part of a transposase and the N-terminal portion of a resolvase were located between the inverted repeats. These open reading frames are most likely the remnants of a defective transposon. A protein with homology to a mercury- resistance regulator was also present within the transposon-like element although no gene encoding for mercury reductase could be indentified.     

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.     

Nucleotide Sequence and Genetic Characterization of the Novel IncQ-like Plasmid pIE1107

The analysis of the complete nucleotide sequence of the small resistance plasmid pIE1107 revealed a close similarity to the well-known IncQ plasmids. Highly conserved replication proteins and nearly identical origins of replication (oriV) suggest equivalent functions in the related replication systems. However, pIE1107 contains two copies of IncQ-oriV-like DNA which are slightly different regarding the iterons. Upon deletion of a silent copy of IncQ-oriV-like DNA the resulting plasmid is fully compatible with IncQ plasmids, indicating that there is no mutual communication between the replication control of the respective replicons. Experiments with clonedoriV DNA strongly suggest that the replication initiation protein of pIE1107 has specialized into the distinct target-iterons of its ownoriV which differs only by a few nucleotides from theoriV of IncQ plasmids. Implications from the apparent highly specific protein–DNA recognition and from the incompatibility properties of pIE1107 for the evolution of a family of compatible, IncQ-like plasmids are discussed.     

Comparative biology of IncQ and IncQ-like plasmids.

Plasmids belonging to Escherichia coli incompatibility group Q are relatively small (approximately 5 to 15 kb) and able to replicate in a remarkably broad range of bacterial hosts. These include gram-positive bacteria such as Brevibacterium and Mycobacterium and gram-negative bacteria such as Agrobacterium, Desulfovibrio, and cyanobacteria. These plasmids are mobilized by several self-transmissible plasmids into an even more diverse range of organisms including yeasts, plants, and animal cells. IncQ plasmids are thus highly promiscuous. Recently, several IncQ-like plasmids have been isolated from bacteria found in environments as diverse as piggery manure and highly acidic commercial mineral biooxidation plants. These IncQ-like plasmids belong to different incompatibility groups but have similar broad-host-range replicons and mobilization properties to the IncQ plasmids. This review covers the ecology, classification, and evolution of IncQ and IncQ-like plasmids.     

Identification and characterization of a native Dichelobacter nodosus plasmid, pDN1

The gram-negative anaerobe Dichelobacter nodosus is the primary causative agent of ovine footrot, a mixed bacterial infection of the hoof. We report here the characterization of a novel native plasmid, pDN1, from D. nodosus. Sequence analysis has revealed that pDN1 has a high degree of similarity to broad-host-range plasmids belonging, or related, to Escherichia coli incompatibility group Q. However, in contrast to these plasmids, pDN1 encodes no antibiotic resistance determinants, lacks genes E and F, and hence is smaller than all previously reported IncQ plasmids. In addition, pDN1 belongs to a different incompatibility group than the IncQ plasmids to which it is related. However, pDN1 does contain the replication and mobilization genes that are responsible for the extremely broad host range characteristic of IncQ plasmids, and derivatives of pDN1 replicate in E. coli. In addition, the mobilization determinants of pDN1 are functional, since derivatives of pDN1 are mobilized by the IncPalpha plasmid RP4 in E. coli.     

A new IncQ plasmid R89S: Properties and genetic organization

The new small (8.18 kb) streptomycin-resistant multicopy plasmid R89S of the Q group incompatibility is described. In contrast to other IncQ plasmids, replication of R89S is dependent on DNA polymerase 1 and proceeds in the absence of de novo protein synthesis. According to our data up to now, the host spectrum of the plasmid R89S is limited to Enterobacteriaceae. A genetic map of the plasmid R89S has been prepared through the construction of deletion and insertion derivatives. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to streptomycin, and the region essential for mobilization of R89S. The origin of vegetative replication has been located within a 0.7-kb fragment. Another region highly homologous to oriV of the plasmid RSF1010, but not functioning as an origin of replication, was localized. Two regions involved in the expression of incompatibility have also been identified. The data from the restriction analyses, DNA-DNA hybridization, and genetic experiments enable us to assume that the plasmid R89S is a naturally occurring recombinant between part of an IncQ plasmid and another narrow host range replicon of unknown incompatibility group.     

Regulation of copy number and stability of phage λ derived pTCλ1 plasmid in the light of the dimer/multimer catastrophe hypothesis

The dimer catastrophe hypothesis has been proposed previously to explain instability of multicopy plasmids whose partitioning is random, contrary to low copy number plasmids which are stably maintained and actively partitioned. Until now, this hypothesis has been investigated using multicopy ColE1 plasmids. However, for more detailed testing of the dimer/multimer catastrophe hypothesis, one should use a plasmid which can be maintained at either low or high copy number and still possesses the same mechanism of replication regulation. Here we used a modified lambda plasmid, pTC lambda 1. The advantage of this plasmid is that it can be maintained at different copy numbers depending on the concentration of an inducer which stimulates the initiation of plasmid replication. Results obtained with this plasmid in recombination proficient and deficient cells generally support the dimer/multimer catastrophe hypothesis, but also suggest some modification in the model.     

Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR.

Primer systems for PCR amplification of different replicon-specific DNA regions were designed on the basis of published sequences for plasmids belonging to the incompatibility (Inc) groups IncP, IncN, IncW, and IncQ. The specificities of these primer systems for the respective Inc groups were tested with a collection of reference plasmids belonging to 21 different Inc groups. Almost all primer systems were found to be highly specific for the reference plasmid for which they were designed. In addition, the primers were tested with plasmids which had previously been grouped by traditional incompatibility testing to the IncN, IncW, IncP, or IncQ group. All IncQ plasmids gave PCR products with the IncQ primer systems tested. However, PCR products were obtained for only some of the IncN, IncP, and IncW group plasmids. Dot blot and Southern blot analyses of the plasmids revealed that PCR-negative plasmids also failed to hybridize with probes derived from the reference plasmids. The results indicated that plasmids assigned to the same Inc group by traditional methods might be partially or completely different from their respective reference plasmids at the DNA level. With a few exceptions, all plasmids related to the reference plasmid at the DNA level also reacted with the primer systems tested. PCR amplification of total DNA extracted directly from different soil and manure slurry samples revealed the prevalence of IncQ- and IncP-specific sequences in several of these samples. In contrast, IncN- and IncW-specific sequences were detected mainly in DNA obtained from manure slurries.     

Plasmid transfer by conjugation in Desulfovibrio desulfuricans

Conjugational transfer of several IncQ plasmids from Escherichia coli to the strictly anaerobic, sulfate-reducing bacterium Desulfovibrio desulfuricans strain G100A was demonstrated. Plasmid DNA from exconjugants was visualized on agarose gels and was used to transform E. coli to the appropriate antibiotic resistances. Neither transfer of IncW and IncP plasmids to strain G100A, nor transfer of any plasmid to D. desulfuricans strain ATCC 27774 was observed. Conjugation of suicide plasmids containing either Tn5 or Tn9 into D. desulfuricans did not result in detectable transposition. Optimal conditions for conjugational transfer and antibiotic resistance levels of strain G100A were examined.     

Plasmids of corynebacteria

Corynebacteria are pleomorphic, asporogenous, Gram-positive bacteria. Included in this group are nonpathogenic soil corynebacteria, which are widely used for the industrial production of amino acids and detergents, and in biotransformation of steroids. Other members of this group are plant and animal pathogens. This review summarizes the current information available about the plasmids of corynebacteria. The emphasis is mainly on the small plasmids, which have been used for construction of vectors for expression of genes in these bacteria. Moreover, considerable information is now available on their nucleotide sequence, gene organization and modes of replication, which would make it possible to further manipulate these plasmids. Other plasmid properties, such as incompatibility and host range, are also discussed. Finally, use of these plasmids as cloning vectors for the expression of heterologous proteins using corynebacteria as hosts is also summarized to highlight the potential of these bacteria as hosts for recombinant DNA.     

Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5alpha by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221

Summary pT181 and pC221 are closely relatedStaphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in therep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping therep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181. The results of complenentation studies conducted using these hybrids have shown that: (i) at the usual level of expression for a wild-type plasmid each Rep protein can initiate replication strictly from its corresponding origin; (ii) when overproduced, the pT181 RepC protein could also act efficiently on the pC221 origin; a functional pT181 origin present in the same host completely prevented this complementation; (iii) in excess, the RepD protein encoded by pC221 could replicate a plasmid carrying the pT181 origin but could not ensure the hereditary stability of such a plasmid in the absence of another active replication system; (iv) when overproduced both RepC and RepD could act on the origin of replication of three other related plasmids pS194, pC223 and pUB112.     

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.     

Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2

Genes encoding toxin-antitoxin proteins are frequently found on plasmids where they serve to stabilize the plasmid within a bacterial population. The toxin-antitoxin proteins do not increase the likelihood of a progeny cell receiving a plasmid but rather function as post-segregational killing mechanisms which decrease the proportion of cells that survive after losing the plasmid. These toxin-antitoxin couples therefore act as plasmid addiction systems. Several new proteic toxin-antitoxin systems have been identified and these systems appear to be ubiquitous on the chromosomes of bacteria and archaea. When placed on plasmids, these chromosomal systems also have the ability to stabilize plasmids and in at least one case, chromosomal- and plasmid-based toxin-antitoxin systems have been shown to interact. Recent findings regarding toxin-antitoxin systems and questions that have arisen as a result of these findings are reviewed.     

The repABC plasmid family

repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in Rhizobiales. Some strains of this bacterial order can contain multiple repABC replicons indicating that this plasmid family includes several incompatibility groups. The replication and stable maintenance of these replicons depend on the presence of a repABC operon. The repABC operons sequenced to date share some general characteristics. All of them contain at least three protein-encoding genes: repA, repB and repC. The first two genes encode proteins involved in plasmid segregation, whereas repC encodes a protein crucial for replication. The origin of replication maps within the repC gene. In contrast, the centromere-like sequence (parS) can be located at various positions in the operon. In this review we will summarize current knowledge about this plasmid family, with special emphasis on their structural diversity and their complex genetic regulation. Finally, we will examine some ideas about their evolutionary origin and trends.     

Structural and functional organization of ColE2 and ColE3 replicons

Summary The complete nucleotide sequences of the 1.5 kb regions of ColE2 and ColE3 plasmids containing the segments sufficient for autonomous replication have been determined. They are quite homologous (greater than 90%), indicating that these two plasmids share common mechanisms of initiation of replication and its regulation. An open reading frame with a coding capacity for a protein of about 300 amino acids is present in both ColE2 and ColE3 and it actually specifies the Rep (for replication) protein, which is the plasmid specific trans-acting factor required for autonomous replication. The amino acid sequences of the Rep proteins of ColE2 and ColE3 are quite homologous (greater than 90%). The cis-acting sites (origins) where replication initiates in the presence of the trans-acting factors consist of 32 bp for ColE2 and 33 bp for ColE3. They are the smallest of all the prokaryotic replication origins so far reported. They are nonhomologous only at two positions, one of which, a deletion of a single nucleotide in ColE2 (or an insertion in ColE3), determines the plasmid specificity in interaction of the origins with the Rep proteins. Both plasmids carry a region with an identical nucleotide sequence and the one in ColE2, the IncA region, has been shown to express incompatibility against both ColE2 and ColE3. These results indicate that these plasmids share a common IncA determinant. A possibility that a small antisense RNA is involved in copy number control and incompatibility (IncA function) was suggested.     

Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

Comparative Biology of Two Natural Variants of the IncQ-2 Family Plasmids,pRAS3.1 and pRAS3.2

Plasmids pRAS3.1 and pRAS3.2 are two closely related, natural variants of the IncQ-2 plasmid family that have identical plasmid backbones except for two differences. Plasmid pRAS3.1 has five 6-bp repeat sequences in the promoter region of the mobB gene and four 22-bp iterons in its oriV region, whereas pRAS3.2 has only four 6-bp repeats and three 22-bp iterons. Plasmid pRAS3.1 was found to have a higher copy number than pRAS3.2, and we show that the extra 6-bp repeat results in an increase in mobB and downstream mobA/repB expression. Placement of repB (primase) behind an arabinose-inducible promoter in trans resulted in an increase in repB expression and an approximately twofold increase in the copy number of plasmids with identical numbers of 22-bp iterons. The pRAS3 plasmids were shown to have a previously unrecognized toxin-antitoxin plasmid stability module within their replicons. The ability of the pRAS3 plasmids to mobilize the oriT regions of two other plasmids of the IncQ-2 family, pTF-FC2 and pTC-F14, suggested that the mobilization proteins pRAS3 are relaxed and can mobilize oriT regions with substantially different sequences. Plasmids pRAS3.1 and pRAS3.2 were highly incompatible with plasmids pTF-FC2 and pTC-F14, and this incompatibility was removed on inactivation of an open reading frame situated downstream of the mobCDE mobilization genes rather than being due to the 22-bp oriV-associated iterons. We propose that the pRAS3 plasmids represent a third, γ incompatibility group within the IncQ-2 family plasmids.Plasmids of the IncQ family are small (<20 kb), have a broad host range, and are highly promiscuous due to their ability to be mobilized very efficiently by self-transmissible plasmids such as the IncP plasmids. They have been divided into two families, IncQ-1 and IncQ-2, based on the amino acid sequence relatedness of their RepA (helicase), RepB (primase), and RepC (DNA-binding) replication proteins and because the mobilization proteins of the two families are unrelated, consisting of three or five genes, respectively (31). IncQ-1 group plasmids include RSF1010 and the near-identical R1162, pDN1, pIE1107, pIE1115, and pIE1130, while IncQ-2 plasmids include pTF-FC2, pTC-F14, and pRAS3.IncQ-2 plasmids pRAS3.1 and pRAS3.2 were isolated in Norway from the fish pathogens Aeromonas salmonicida subsp. salmonicida and atypical A. salmonicida, respectively, while investigating plasmids that conferred resistance to tetracycline (21). The two plasmids encode identical replication and mobilization proteins, with the most important differences in the plasmid backbone being that pRAS3.1 has four 22-bp iterons in its oriV region and five 6-bp repeat sequences upstream of its mobB gene, whereas pRAS3.2 has only three iterons and four 6-bp repeat sequences. No biological studies were carried out in the initial report of the pRAS3 plasmids. As a contribution to our studies on the evolution of IncQ plasmids, our longer-term aim is to address the question of why two natural versions of the plasmid exist. Here we report on the major differences in the biology of the two plasmids. In addition, we discovered the presence of repC and mobB genes that were not detected when the sequence of pRAS3 plasmids was previously reported. We also discovered a putative toxin-antitoxin (TA) postsegregational system different from that found in other members of the IncQ plasmids and tested it for functionality.The IncQ-1 plasmids are subdivided into incompatibility groups α, β, and γ, (31), whereas the IncQ-2 plasmids are subdivided into two incompatibility groups, α and β (14). In this work we also report on the incompatibility between the pRAS3 plasmids and other members of the IncQ-2 plasmid family as well as the IncQ-1 family plasmids. Furthermore, we compare the functional relatedness of the pRAS3 mobilization system with that of previously studied IncQ-2 plasmids.