Identification of cistrons involved in conjugal transfer of narrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa.

The development of a transductional method for complementation tests between transfer-deficient mutants of the narrow-host-range R plasmic R91-5 of Pseudomonas aeruginosa has allowed the indentification of cistrons involved in the conjugal transfer of this plasmid. Complementation tests performed between transfer-deficient mutants characterized phenotypically with respect to sensitivity to donor-specific phage, ability to inhibit the replication of phage G101, and expression of entry-exclusion has identified a minimum of 10 transfer cistrons. Although most mutagen-induced mutants were relatively heterogeneous and appeared to be affected in a single cistron only, a high proportion of mutants isolated after selection for donor-specific phage resistance had deletions but always included tra Y. Mutants selected directly on the basis of transfer deficiency which also became donor-specific phage resistant fell into all 10 cistrons, suggesting that many R91-5 transfer cistrons are concerned with the synthesis of sex pili and other surface structures necessary for conjugal transfer. Conversely, most retaining donor-specific phage sensitivity belonged to one cistron, whereas transfer-deficient mutants which had also lost the ability to inhibit the replication of phage G101 comprised four cistrons.     

Insertion mutations in the promiscuous IncP-1 plasmid R18 which affect its host range between Pseudomonas species

Fifty-one host range mutants of the promiscuous plasmid R18 were isolated by Tn7 insertion mutagenesis by using Pseudomonas aeruginosa as the permissive, and P. stutzeri as the nonpermissive, host. Endonuclease cleavage mapping of 40/51 mutants showed that 37 mutations mapped to kilobase coordinates 40.3-43.8 in the two overlapping genes encoding plasmid DNA primase. Thus by this procedure it has been possible readily to isolate a large number of primase mutants. The majority of these mutations mapped to the overlapping DNA whereas a few also mapped to the nonoverlap region encoding the larger 118-kDa polypeptide. Among these mutants were four which had long deletions within the overlapping segment and extending to varying lengths anticlockwise of it. The genetic defect in these mutants has been correlated with greatly reduced in vitro primase enzyme activity. The primase mutations drastically affected the mutant's ability to mobilize a nonconjugative, wide-host-range IncP-4(Q) plasmid from P. aeruginosa to P. stutzeri although mobilization within P. aeruginosa was affected to a lesser degree. Other insertion mutations were mapped to the regions of plasmid origin of transfer (oriT) and origin of replication (oriV), but their physical location was different to previously identified similar mutations obtained using Escherichia coli as the nonpermissive host. Their physically distinct locations were correlated with differences in their transmissibility from P. aeruginosa into enteric bacterial species and into other Pseudomonas species.     

Deletion Map of the Escherichia coli K-12 Sex Factor F: the Order of Eleven Transfer Cistrons

A series of Hfr deletion mutants was isolated. These mutants contain deletions which extend from a lambda prophage into an Flac which is integrated into the gal operon. Transfer-deficient deletion mutants were found to fall into four different phenotypic groups when tested for male- and female-specific phage resistance. Conjugational and transductional complementation tests with Flac point mutants deficient in transfer (tra(-)) were performed, and the order of 11 tra cistrons was determined. The tra genes are all located between an F gene for the inhibition of female-specific phages and the transposed lac operon originally carried by the Flac. The order of genes in the Hfr studied was established to be: proC... phi(II) (R)... traJ traA traE traK traB traC traF traH traG traD traI...lac...attlambda...bio.     

Characterization of the functional sites in the oriT region involved in DNA transfer promoted by sex factor plasmid R100.

We have previously identified three sites, named sbi, ihfA, and sbyA, specifically recognized or bound by the TraI, IHF, and TraY proteins, respectively; these sites are involved in nicking at the origin of transfer, oriT, of plasmid R100. In the region next to these sites, there exists the sbm region, which consists of four sites, sbmA, sbmB, sbmC, and sbmD; this region is specifically bound by the TraM protein, which is required for DNA transfer. Between sbmB and sbmC in this region, there exists another IHF-binding site, ihfB. The region containing all of these sites is located in the proximity of the tra region and is referred to as the oriT region. To determine whether these sites are important for DNA transfer in vivo, we constructed plasmids with various mutations in the oriT region and tested their mobilization in the presence of R100-1, a transfer-proficient mutant of R100. Plasmids with either deletions in the sbi-ihfA-sbyA region or substitution mutations introduced into each specific site in this region were mobilized at a greatly reduced frequency, showing that all of these sites are essential for DNA transfer. By binding to ihfA, IHF, which is known to bend DNA, may be involved in the formation of a complex (which may be called oriT-some) consisting of TraI, IHF, and TraY that efficiently introduces a nick at oriT. Plasmids with either deletions in the sbm-ihfB region or substitution mutations introduced into each specific site in this region were mobilized at a reduced frequency, showing that this region is also important for DNA transfer. By binding to ihfB, IHF may also be involved in the formation of another complex (which may be called the TraM-IHF complex) consisting of TraM and IHF that ensures DNA transfer with a high level of efficiency. Several-base-pair insertions into the positions between sbyA and sbmA affected the frequency of transfer in a manner dependent upon the number of base pairs, indicating that the phasing between sbyA and sbmA is important. This in turn suggests that both oriT-some and the TraM-IHF complex should be in an appropriate position spatially to facilitate DNA transfer.     

Transfer-deficient mutants of the narrow-host-range plasmid R91-5 of Pseudomonas aeruginosa.

Three methods have been successful in the isolation of transfer-deficient mutants of the narrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa: (i) selection for donor-specific phage resistance; (ii) direct screening after mutagenic treatment with either ethyl methane sulfonate or N-methyl-N'-nitro-N-nitrosoguanidine; (iii) in vitro mutagenesis of plasmid DNA by hydroxylamine followed by transformation and direct screening. The majority of transfer-deficient mutants were donor-specific phage resistant, supporting the view that sex pili and other surface components are essential for conjugal transfer (since the phages PRD1 and PR4 adsorb to these sites). Some of the transfer-deficient mutants were also unable to inhibit the replication of phage G101 or lost entry exclusion or both phenotypes. The ability to revert these pleiotropic mutants to wild type implicates the latter two functions in R91-5 transfer. Suppressor mutations in P. aeruginosa enabled the detection of suppressor-sensitive, transfer-deficient mutants. Such mutants should prove useful in conjugational complementation tests for the identification of the transfer cistrons of R91-5.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.     

Cloning of the pif region of the F sex factor and identification of a pif protein product

This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages. We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F. All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases. A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103. A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103. Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein.     

Genetic map of an octopine TI-plasmid.

Several deletion mutants of an octopine TI-plasmid were mapped by digestion with the restriction enzyme Sma I. The T region, as it is defined on the B6-806 plasmid, does not appear to be an essential area for tumour induction on the plasmid of Ach5. The genes for octopine breakdown, plasmid transfer, and the replicator were roughly localized. The possibility of using mutants with large deletions as a cloning vehicle in Agrobacterium tumefaciens is discussed.     

Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC.

Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA. This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation. This region contains three genes, trwA, trwB, and trwC. By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination. Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination. TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process. Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases. This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required. This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.     

Deletion analysis of the F plasmid oriT locus.

Functional domains of the Escherichia coli F plasmid oriT locus were identified by deletion analysis. DNA sequences required for nicking or transfer were revealed by cloning deleted segments of oriT into otherwise nonmobilizable pUC8 vectors and testing for their ability to promote transfer or to be nicked when tra operon functions were provided in trans. Removal of DNA sequences to the right of the central A + T-rich region (i.e., from the direction of traM) did not affect the susceptibility of oriT to nicking functions; however, transfer efficiency for oriT segments deleted from the right was progressively reduced over an 80- to 100-bp interval. Deletions extending toward the oriT nick site from the left did not affect the frequency of transfer if deletion endpoints lay at least 22 bp away from the nick site. Deletions or insertions in the central, A + T-rich region caused periodic variation in transfer efficiency, indicating that phase relationships between nicking and transfer domains of oriT must be preserved for full oriT function. These data show that the F oriT locus is extensive, with domains that individually contribute to transfer, nicking, and overall structure.     

Genetic organization of plasmid R1162 DNA involved in conjugative mobilization.

DNA involved in the mobilization of broad-host-range plasmid R1162 was localized to a region of 2.7 kilobases within coordinates 3.4 to 6.1 kilobases on the R1162 map. By examining the transfer properties of plasmids containing cloned fragments of DNA from within this region, we showed that at least four trans-active products and a cis-active site (oriT) were involved in mobilization. A cloned DNA fragment of 155 base pairs was capable of providing full oriT activity. This fragment was located within 600 base pairs of DNA containing the origin of replication of R1162, and its nucleotide sequence and that of neighboring DNA were determined. Activation of oriT required R1162-encoded, trans-acting products. Deletions which resulted in the loss of one or more of these had a variable effect on transfer efficiency and indicated the presence of both essential and nonessential Mob products. Regions encoding these products flanked oriT and in one case appeared to overlap a gene essential for plasmid replication. The implications of these findings with respect to the broad host range of R1162 are discussed.     

Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes.

The conjugation system of the IncP alpha plasmid RK2/RP4 is encoded by transfer regions designated Tra1, Tra2, and Tra3. The Tra1 core region, cloned on plasmid pDG4 delta 22, consists of the origin of transfer (oriT) and 2.6 kilobases of flanking DNA providing IncP alpha plasmid-specific functions that allow pDG4 delta 22 to be mobilized by the heterologous IncP beta plasmid R751. Tn5 insertions in pDG4 delta 22 define a minimal 2.2-kilobase region required for plasmid-specific transfer of oriT. The Tra1 core contains the traJ and traK genes as well as an 18-kilodalton open reading frame downstream of traJ. The traJ and traK genes were shown to be required for transfer by complementation of inserts within these genes. Genetic evidence for the role of the 18-kilodalton open reading frame in transfer was obtained, although this protein has not been detected in cell lysates. These studies indicate that at least three transfer proteins are involved in plasmid-specific interactions at oriT.     

Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.     

Identification of functional mob regions in Rhizobium etli: evidence for self-transmissibility of the symbiotic plasmid pRetCFN42d

An approach originally designed to identify functional origins of conjugative transfer (oriT or mob) in a bacterial genome (J. A. Herrera-Cervera, J. M. Sanjuán-Pinilla, J. Olivares, and J. Sanjuán, J. Bacteriol. 180:4583-4590, 1998) was modified to improve its reliability and prevent selection of undesired false mob clones. By following this modified approach, we were able to identify two functional mob regions in the genome of Rhizobium etli CFN42. One corresponds to the recently characterized transfer region of the nonsymbiotic, self-transmissible plasmid pRetCFN42a (C. Tun-Garrido, P. Bustos, V. González, and S. Brom, J. Bacteriol. 185:1681-1692, 2003), whereas the second mob region belongs to the symbiotic plasmid pRetCFN42d. The new transfer region identified contains a putative oriT and a typical conjugative (tra) gene cluster organization. Although pRetCFN42d had not previously been shown to be self-transmissible, mobilization of cosmids containing this tra region required the presence of a wild-type pRetCFN42d in the donor cell; the presence of multiple copies of this mob region in CFN42 also promoted conjugal transfer of the Sym plasmid pRetCFN42d. The overexpression of a small open reading frame, named yp028, located downstream of the putative relaxase gene traA, appeared to be responsible for promoting the conjugal transfer of the R. etli pSym under laboratory conditions. This yp028-dependent conjugal transfer required a wild-type pRetCFN42d traA gene. Our results suggest for the first time that the R. etli symbiotic plasmid is self-transmissible and that its transfer is subject to regulation. In wild-type CFN42, pRetCFN42d tra gene expression appears to be insufficient to promote plasmid transfer under standard laboratory conditions; gene yp028 may play some role in the activation of conjugal transfer in response to as-yet-unknown environmental conditions.     

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.     

Identification and cloning of the conjugative transfer region of Staphylococcus aureus plasmid pGO1.

The conjugative transfer (tra) genes of a 52-kilobase (kb) staphylococcal plasmid, pGO1, were localized by deletion analysis and transposon insertional inactivation. All transfer-defective (Tra-) deletions and Tn551 or Tn917 transposon insertions occurred within a 14.5-kb BglII fragment. Deletions and insertions outside this fragment all left the plasmid transfer proficient (Tra+). The tra region was found to be flanked by directly repeated DNA sequences, approximately 900 base pairs in length, at either end. Clones containing the 14.5-kb BglII fragment (pGO200) and subclones from this fragment were constructed in Escherichia coli on shuttle plasmids and introduced into Staphylococcus aureus protoplasts. Protoplasts could not be transformed with pGO200E (pGO200 on the staphylococcal replicon, pE194) or subclones containing DNA at one end of the tra fragment unless pGO1 or specific cloned tra DNA fragments were present in the recipient cell. However, once stabilized by sequences present on a second replicon, each tra fragment could be successfully introduced alone into other plasmid-free S. aureus recipients by conjugative mobilization or transduction. In this manner, two clones containing overlapping fragments comprising the entire 14.5-kb BglII fragment were shown to complement each other. The low-frequency transfer resulted in transconjugants containing one clone intact, deletions of that clone, and recombinants of the two clones. The resulting recombinant plasmid (pGO220), which regenerated the tra region intact on a single replicon, transferred at frequencies comparable to those of pGO1. Thus, all the genes necessary and sufficient for conjugative transfer of pGO1 are contained within a 14.5-kb region of DNA.     

Location of the traS- and finO-type genes and of the oriT region of plasmid R6K

The regions of B6K DNA corresponding to oriT, finO and traS are mapped using a number of hybrid plasmids and deletion mutants pAS3::Tn5, pAS3::Tn7 and pAS3::Tn9 obtained in vitro after treatment with restriction endonucleases EcoRI and BamHI. FinO- and traS-like genes were mapped in the regions of 10 and 25,0-25,4 MD, respectively, oriT being situated in the region of 4,6-4,7 Md.