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Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2.

ABSTRACT: Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.

SUBMITTER: van Zyl LJ

PROVIDER: S-EPMC225039 | biostudies-literature | 2003 Oct

REPOSITORIES: biostudies-literature

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Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2.

van Zyl Leonardo J LJ Deane Shelly M SM Rawlings Douglas E DE

Journal of bacteriology 20031001 20

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second examp ...[more]

PMID: 14526022

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Project description:A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.

Project description:The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.

Dataset Information

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2.

Publications

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2.

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