Site-directed mutations in the relaxase operon of RP4.

Mutations were constructed by site-directed mutagenesis in the relaxase operon of the broad-host-range plasmid RP4. The mutations were constructed in smaller plasmids, recombined into the 60-kb RP4 plasmid, and tested for their ability to transfer. The relaxase operon contains the transfer genes traJ, traH, and traI, which are involved in nicking at the transfer origin to generate the single strand destined to be transferred to the recipient cell. In the first mutant, the C terminus of TraI was truncated, leaving TraH intact. This mutant decreased transfer by approximately 500-fold in Escherichia coli, and the traI mutation could be complemented by a wild-type copy of traI in trans in the donor. The traI mutation similarly decreased transfer between a variety of gram-negative bacteria. A site-specific mutation was made by the polymerase chain reaction-based unique-site mutagenesis procedure to alter the start site of traH. This mutation had no effect on intraspecific E. coli transfer but reduced transfer by up to sevenfold for some gram-negative bacteria. The traH mutation had no effect on plasmid stability. Thus, neither TraH nor the C terminus of TraI is required for conjugative transfer, but both increase mating efficiency in some hosts.     

Analysis of Escherichia coli K12 F factor transfer genes: traQ, trbA, and trbB

The genes that encode the transfer properties of plasmid F, the fertility factor of Escherichia coli K12, are known to be clustered over a large, 33.3-kb segment of F DNA. As the central segment of the transfer region has not previously been well characterized, we constructed a detailed restriction map of the large F EcoRI DNA fragment, fl, and isolated a series of plasmid derivatives that carry various overlapping segments of this F tra operon DNA. We also analyzed the protein products of those clones that carried DNA segments extending over the region between traF and traH. This region was known to include traQ, a gene required for efficient conversion of the direct product of traA to the 7000-Da pilin polypeptide. We identified the traQ product as a polypeptide that migrates as a 12,500-Da protein on sodium dodecyl sulfate-polyacrylamide gels. We also detected the products of two other new genes that we have named trbA and trbB. These polypeptides migrate with apparent molecular weights of 14,200 and 18,400, respectively. Analysis of plasmid deletion derivatives that we constructed in vitro shows that these genes map in the order traF trbA traQ trbB traH. The presence of a plasmid carrying a small 0.43-kb fragment that expressed only the 12,500 traQ product caused the traA product of a co-resident compatible plasmid to be converted to the 7000-Da pilin polypeptide, demonstrating that TraQ is the only tra operon product required for this step of F-pilin biosynthesis.     

A new plasmid pBS1001 with broad range of hosts

A new broad host-range plasmid capable of conjugative transfer has been isolated and characterized. The plasmid has the high frequency of conjugation transfer, is capable of conjugative transfer mobilization of nonconjugative plasmids, carries no known phenotypic markers. The plasmid demonstrates the specific interaction with the plasmids of P incompatibility group. The comparatively small size of the plasmid permits one to use it efficiently for comparative study of organization of the broad host range plasmids.     

Functional and mutational analysis of conjugative transfer region 1 (Tra1) from the IncHI1 plasmid R27

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.     

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone.

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.     

Genetic analysis of insertion mutations of the promiscuous IncP-1 plasmid R18 mapping near oriT which affect its host range

Transposon Tn7 insertion mutations of the promiscuous IncP-1 plasmid R18 which affect its conjugational transmissibility from Pseudomonas aeruginosa to Escherichia coli C, a strain of E. coli K12, Salmonella typhimurium and P. maltophilia have been mapped physically. They map to coordinate 53.5 kb in the Tral region of the plasmid. An 800-bp fragment mapping between R18 coordinates 52.85 and 53.65 kb, which complemented the host range defect of the mutants when tested with E. coli C as recipient, has been identified. However, complementation occurred only when the 800-bp cloned fragment was provided in the E. coli C recipient but not when situated in the P. aeruginosa donor. It is concluded that a trans-acting gene product of R18 is required, in the transcipient, for conjugative DNA metabolism during, or immediately following, the conjugational transfer of this plasmid between certain donor and recipient hosts.     

Investigating the impact of bisphosphonates and structurally related compounds on bacteria containing conjugative plasmids

Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in Escherichia coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli cells. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation.     

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.     

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.     

Vibrio cholerae hybrid sex factor that contains ampicillin transposon Tn1.

The ampicillin resistance transposon Tn1 was translocated from the R plasmid RP4 to the Vibrio cholerae conjugative plasmid, P. The hybrid sex factor P::Tn1 was highly transmissible and expressed the biological activities of the P factor. In addition, P::Tn1 facilitated transfer of RP4 to V. cholerae recipients. Physical studies of P::Tn1 indicated that the Tn1 transposon was added to the otherwise unaltered P plasmid.     

Molecular analysis of the F plasmid traVR region: traV encodes a lipoprotein.

The nucleotide sequences of the conjugative F plasmid transfer region genes, traV and traR, have been determined. The deduced amino acid sequence of TraV indicated that it may be a lipoprotein; this was confirmed by examining the effect of globomycin on traV-encoded polypeptides synthesized in minicells. An open reading frame that may represent a previously undetected transfer gene, now designated trbG, was identified immediately upstream of traV. The deduced product of traR was found to share amino acid similarity with proteins from the bacteriophages 186 and P2 and with the dosage-dependent dnaK suppressor DksA.     

Preparation of bacteria--degraders of alkylnaphthalenesulfonate by a molecular breeding method

Transconjugant strain of bacteria degraders of alkylnaphthalenesulphonate(ANS)--P Pseudomonas alcaligenes TR (NPL-41) was obtained under conditions of ANS selective pressure and flow cultivation of bacteria destructors of alkylbenzene sulphonate (ABS) P. alcaligenes TR (pABS) and naphthalene P. putida BS438 (NPL-41) and their immobilization on an inert carrier. Transconjugant P. alcaligenes TR(NPL-41) was prepared by means of conjugative transfer of plasmid NPL-41 from P. putida BS438 to P. alcaligenes TR. The check out of transconjugant under conditions of flow cultivation showed their ability to degradation of ANS. Investigation of the stability of plasmid NPL-41 in the transconjugant P. alcaligenes TR(NPL-41), obtained by molecular breeding under conditions of flow cultivation and by conjugative transfer, showed the transconjugant prepared by the former method to be more stable.     

Mechanism of plasmid ColEl and pMB-9 mobilization by plasmid F'lac+ in Escherichia coli K-12]

The genetic control and mechanism of mobilization of the non-conjugative plasmids ColE1 and pMB-9 by the conjugative plasmids was orived to be recA-independent process in contrast to the mobilization of the chromosomal marker pro. Acridine orange and ethidium bromide curing data together with the results of electrophoretic analysis of plasmid DNA suggest that the plasmids F' lac+ and pMB-9 as well as F' lac+ and ColE1 remain autonomous after their contransfer to recipient cells. These data argue in favour of non-recombinational nature of the plasmid mobilization process. The possibility of transmission of a non-conjugative plasmid without transmission of a conjugative one from the donor strain carrying both plasmids was established. The results obtained are discussed with respect to the hypothesis on the effect of diffusible products encoded by the conjugative plasmid and required of the mobilization of the non-conjugative plasmid.     

Conjugative R-plasmid of facultative methylotrophic bacteria Pseudomonas sp. M

50 Md conjugative plasmid, designated pM3, has been found in the cells from natural isolates of Pseudomonas sp M. The plasmid determines the resistance to tetracycline and streptomycin and is capable of conjugative transfer between the cells of Pseudomonas and Escherichia coli. The conjugative derivatives of pM3 deleted for 14 Md of molecular mass were isolated after acridine dyes treatment of cells harbouring plasmid pM3. The discovered plasmid was not shown to belong to IncP1 incompatibility group.     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

The kilE locus of promiscuous IncP alpha plasmid RK2 is required for stable maintenance in Pseudomonas aeruginosa.

Eight coordinately regulated operons constitute the kor regulon of the IncP alpha plasmid RK2. Three operons specify functions required for replication initiation, conjugative transfer, and control of gene expression. The functions of the other operons, including those of the four coregulated operons that compose the kilA, kilC, and kilE loci, have not been determined. Here, we present the first evidence that a kil determinant is involved in IncP plasmid maintenance. Elevation of KorC levels specifically to reduce the expression of the KorC-regulated kilC and kilE operons severely affected the maintenance of both the IncP alpha plasmid RK2lac and the IncP beta plasmid R751 in Pseudomonas aeruginosa but had little effect on plasmid maintenance in Escherichia coli. Precise deletion of the two kilE operons from RK2lac was achieved with the VEX mutagenesis system for large genomes. The resulting plasmid showed significant loss of stability in P. aeruginosa only. The defect could be complemented by reintroduction of kilE at a different position on the plasmid. The instability of the RK2lac delta kilE mutant did not result from a reduction in average plasmid copy number, reduced expression of kilC, decreased conjugative transfer, or loss of the korE regulator. We found that both the par and kilE loci are required for full stability of RK2lac in P. aeruginosa and that the par and kilE functions act independently. These results demonstrate a critical role for the kilE locus in the stable inheritance of RK2 in P. aeruginosa.     

Complete nucleotide sequence of a cryptic plasmid from the marine bacterium Vibrio splendidus and identification of open reading frames

A 2.3-kb replication-proficient fragment was previously obtained from a cryptic plasmid (pPS41) isolated from a marine Vibrio splendidus isolate (P. A. Sobecky, T. J. Mincer, M. C. Chang, A. Toukdarian, and D. R. Helinski, 1998, Appl. Environ. Microbiol. 64, 2822-2830). Analysis of the complete nucleotide sequence of plasmid pPS41 revealed two additional open reading frames (ORFs). Analysis of ORF-1 revealed that its translated product has 125 amino acids with a predicted MW of 16,978 and ORF-2 encodes a putative protein of 151 amino acids with a predicted MW of 19,802. The ORF-2 encoded protein showed 31 to 35% sequence homology to proteins identified to have a role in plasmid mobilization. These proteins are encoded on plasmids found in Escherichia coli and Pasteurella multocida. Plasmid pPS41 could be mobilized by a conjugative plasmid at frequencies of 1 x 10(-2) to 2 x 10(-2).     

Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens

Clostridium perfringens causes fatal human infections, such as gas gangrene, as well as gastrointestinal diseases in both humans and animals. Detailed molecular analysis of the tetracycline resistance plasmid pCW3 from C. perfringens has shown that it represents the prototype of a unique family of conjugative antibiotic resistance and virulence plasmids. We have identified the pCW3 replication region by deletion and transposon mutagenesis and showed that the essential rep gene encoded a basic protein with no similarity to any known plasmid replication proteins. An 11-gene conjugation locus containing 5 genes that encoded putative proteins with similarity to proteins from the conjugative transposon Tn916 was identified, although the genes' genetic arrangements were different. Functional genetic studies demonstrated that two of the genes in this transfer clostridial plasmid (tcp) locus, tcpF and tcpH, were essential for the conjugative transfer of pCW3, and comparative analysis confirmed that the tcp locus was not confined to pCW3. The conjugation region was present on all known conjugative plasmids from C. perfringens, including an enterotoxin plasmid and other toxin plasmids. These results have significant implications for plasmid evolution, as they provide evidence that a nonreplicating Tn916-like element can evolve to become the conjugation locus of replicating plasmids that carry major virulence genes or antibiotic resistance determinants.