Cloning the Tra1 region of RP1

Watson, John M.; Schmidt, Londa; Willetts, Neil

doi:10.1016/0147-619x(80)90007-4

Cited by 20 publications

(4 citation statements)

References 19 publications

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“…Furthermore, it can be concluded that functions essential for the mating pair formation system are encoded not only in Tra2 but also in Tral. These results confirm earlier observations that certain insertion mutations in Tral and Tra2 result in resistance to donor-specific phage (4,13,59). To determine which of the gene products encoded by Tral or Tra2 are essential for conjugative transfer of RP4, the mobilization of RSF1010, and the propagation of donor-specific bacteriophages, nonpolar mutants of the individual tra genes have to be constructed and analyzed.…”

Section: Materuils and Methodssupporting

confidence: 81%

Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans

et al. 1992

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We constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions Tral and Tra2 of the broad-host-range IncP plasmid RP4. In this system, the plasmid containing the Tral region with the origin of transfer (oriT) was transferred, whereas additional functions essential for the conjugative process were provided from the Tra2 plasmid in trans. The Tra2 region, as determined for matings between Escherichia coli cells, maps between coordinates 18.03 and 29.26 kb of the RP4 standard map. The section of Tra2 required for mobilization of the plasmid RSF1010 (IncQ) and the propagation of bacteriophages Pf3 and PRD1 appears to be the same as that needed for RP4 transfer. Tra2 regions of RP4 (IncPa) and R751 (IncPj3) are interchangeable, facilitating mobilization of the plasmid carrying the RP4 Tral region. The transfer frequencies of both systems are similar. Transcription of Tra2 proceeds clockwise relative to the standard map of RP4 and is probably initiated at a promoter region located upstream of trbB (kilB). From this promoter region the trfA operon and the Tra2 operon are likely to be transcribed divergently. A second potential promoter has been located immediately upstream of trbB (kiIB). Plasmids encoding the functional Tra2 region can only be maintained stably in host cells in the presence of the RP4 regulation region carrying the korA-korB operon or part of it. This indicates the involvement of RP4 key regulatory functions that apparently are active not only in the control of replication but also in conjugation.The conjugative transfer system of the promiscuous plasmid RP4 consists of two distinct regions, Tral and Tra2, separated by the Par (partitioning)/Mrs (multimer resolution system) region, the fiwA locus, IS8, and the kanamycin resistance gene (aphA). Tral, containing the origin of transfer (oriT), encodes functions involved in generating the single strand to be transferred and also includes the primase genes (59; for reviews see references 19 and 60). However, functions encoded by Tra2 have not yet been characterized extensively. Genetic approaches by transposon mutagenesis and complementation studies located the Tra2 region between the genes trbB (kiIB) andfiwA (3,4,37,51 acting replication function), encoding the replication protein and the Par/Mrs region, specifying a multimer resolution system (17,41). Plasmids containing the minimal functional Tra2 region could only be maintained stably when the korA-korB operon was present in trans, implying that KorA and KorB are involved in regulating expression of the transfer loci of Tra2. MATERUILS AND METHODSStrains, phages, and plasmids. E. coli HB101 (8) and S17-1 (47) were used as hosts for plasmids, and the nalidixic acid-resistant strain HB101 Nxr was used as a recipient for filter matings. Cells were grown in YT medium (33) buffered with 25 mM 3-(N-morpholino)propanesulfonic acid (sodium salt, pH 8.0) and supplemented with 0.1% glucose and 25 ,ug of thiamine-hydrochloride per ml. When appropriate, antibiotics...

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Section: Materuils and Methodssupporting

confidence: 81%

Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans

et al. 1992

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“…Former investigations indicated that functions in both transfer regions, Tral and Tra2, are necessary for IncQ mobilization (36) and phage propagation (4,13,62,74). Our complementation and deletion analysis has identified traF and traG as the only Tral components required in trans with Tra2, for expression of a Mob' phenotype, whereas for phage growth, traF in addition to trb genes is sufficient.…”

Section: Methodsmentioning

confidence: 96%

The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation

et al. 1993

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Transfer functions of the conjugative plasmid RP4 (IncPot) are distributed among distinct regions of the genome, designated Tral and Tra2. By deletion analyses, we determined the limits of the Tral region, essential for intraspecific Escherichia coli matings. The Tral core region encompasses approximately 5.8 kb, including the genes traF, -G, -H, -I, -J, and -K as well as the origin of transfer. The traM gene product, however, is not absolutely required for conjugation but significantly increases transfer efficiency. To determine the transfer phenotype of genes encoded by the Tra2 core region, we generated a series of defined Tra2 mutants. This revealed that at least trbB, -C, -E, -G, and -L are essential for RP4 conjugation. To classify these transfer functions as components of the DNA transfer and replication (Dtr) or of the mating pair fonnation (Mpf) system, we analyzed the corresponding derivatives with respect to mobilization of IncQ plasmids and donor-specific phage propagation. We found that all of the Tra2 genes listed above and the traG and traF genes of Tral are required for RSF1010 mobilization. Expression oftraF from Tral in conjunction with the Tra2 core was sufficient for phage propagation. This implies that the TraG protein is not directly involved in pilus formation and potentially connects the relaxosome with proteins enabling the membrane passage of the DNA. The proposed roles of the RP4 transfer gene products are discussed in the context of virulence functions encoded by the evolutionarily related Ti T-DNA transfer system of agrobacteria.The study of conjugative DNA transfer of IncP plasmids gains increasing interest, since transfer of these plasmids to a wide range of remotely related microorganisms (for review, see references 21, 23, 65, and 76), even including yeasts (25), was observed. In addition, several findings provide evidence for a close relationship between RP4-mediated bacterial conjugation and T-DNA transfer from agrobacteria to plant cells (37,39,49,72,81). These similarities are underscored by recent data demonstrating that theAgrobacterium tumefaciens pTi Vir region can mobilize the small non-self-transmissible IncQ plasmid RSF1010 among agrobacteria (7) in a manner resembling the mobilization of these plasmids by IncP plasmids (16,78). However, it is noteworthy that mobilization frequencies obtained by the Ti system are extremely low.Functions responsible for RP4 transfer are encoded by two distinct regions of the genome, designated Tral and Tra2. The Tral region encompasses the origin of transfer (oriT) as well as functions responsible for the cleaving-joining process at the nick site (traI, -J, and -K; for review, see references 23 and 76). The traI and traJ gene products are required for relaxosome formation as well as for site-and strand-specific nicking at oriT, by which Tral becomes covalently associated with the 5' end of the DNA strand destined for transfer to the recipient cell (51). traK and traJ are specificity determinants on the basis of the observation that the...

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“…The third gene in this segment, i.e., traE (61) (Fig. 2), may also be a pilus gene, given that Watson et al (53) reported two Dps-cistrons in Tral in addition to a pleiotropic cistron.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of RP1 Tra1 cistrons involved in pilus function and plasmid mobilization

Fong

Stanisich

1993

J Bacteriol

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Transfer-defective mutants of the Tral region of RP1 were isolated. Complementation studies involving stable heterozygotes combined with the mapping of TnS insertion mutations revealed two pilus cistrons, pi4and pilB, at positions 46.9 to 48.2 kb and 46.0 to 46.4 kb, respectively. All pilB mutants were Dps-(i.e., resistant to donor-specific phages PR4 and PRR1), whereas piL4 mutants were Dps-(promoter-proximal mutations), Dps+'-(sensitive only to PR4 [more centrally located mutations]), or Dps' (sensitive to both phages [promoter-distal mutations]). The correlation between the site mutated and the Dps phenotype, together with the finding that certain Dps+ piL4 mutants continued to mobilize nonconjugative plasmids, suggested that piL4 is bifunctional, contributing both to pilus function (at the promoter-proximal end) and to RP1 mobilization. It was also shown that the 43.5-to 49.5-kb region that includes pilA and pilB encodes all of the Tral pilus functions required for propagation of donor-specific phages and hence, probably, for pili that are active in conjugation. Finally, three cistrons that specifically affect RP1 mobilization were identified. Two of these, mobA and mobB, occur immediately anticlockwise to oriT and probably correspond to the traJ and traI genes characterized by other workers. The third cistron, mobC, occurs clockwise to oriT and may be a new mobilization gene, since its function can be substituted by IncPp plasmids, a feature different from that of the traK mobilization gene which occurs in the same region but is RP1 specific. None of the mob cistrons was required for mobilization of nonconjugative plasmids, except for mobB, which was required by pVS99.The broad-host-range IncPa plasmid RP1 (60 kb; synonymous with R18, RP4, and RK2) carries two regions (Tral and Tra2) that encode conjugal functions (22,37,45

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Cloning the Tra1 region of RP1

Cited by 20 publications

References 19 publications

Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans

Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans

The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation

Identification and characterization of RP1 Tra1 cistrons involved in pilus function and plasmid mobilization

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