RepFIC, a basic replicon of IncFI plasmids that has homology with a basic replicon of IncFII plasmids
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Replicon
Homology
T-DNA Binary system
Autonomously replicating sequence
Replicon
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ABSTRACT A moderately thermophilic (45 to 50°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.
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A cryptic plasmid, pBAA1, was identified in an industrial Bacillus strain. The plasmid is 6.8 kilobases in size and is present in cells at a copy number of approximately 5 per chromosome equivalent. The plasmid has been maintained under industrial fermentation conditions without apparent selective pressure and so is assumed to be partition proficient. The minimal replicon was localized to a 1.4-kilobase fragment which also contains the functions required for copy number control. The very low level of segregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small gram-positive plasmids which replicate by a rolling cycle-type mechanism. A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.
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T-DNA Binary system
Rolling circle replication
Autonomously replicating sequence
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Replicon
T-DNA Binary system
Autonomously replicating sequence
Rolling circle replication
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Abstract Due to their relatively small size and the lack of housekeeping genes, bacterial plasmids are very convenient models for studying DNA replication. So, for a long time, they had been intensively studied in this regard. Unfortunately, only a limited number of model plasmid replication systems were analyzed in detail. In the era of high-throughput DNA sequencing, we are faced with an increasing gap in our knowledge of bacterial plasmid replication and a rapidly growing number of deposited new plasmid genome sequences. For this reason, we decided to investigate the replication system of the pIGMS31 plasmid, a representative of the newly described pHW126-like plasmids family. They are small replicons isolated from different clinical and environmental strains of Gamma proteobacteria. Whole shares unique replication modules with no significant similarities to known model plasmid replication systems. In this study, we identified and characterized the basic elements of the pIGMS31 replication module. Studies on regulatory mechanisms of replication initiation of this plasmid as well as on pIGMS31 replication mode were also performed. We revealed that the pIGMS31 replication module is composed of elements typical for both theta and rolling circle replicons. This mosaic structure is reflected in the unique course of replication of this plasmid, with both modes of replication. What is more, our results led us to conclude that pHW126-like plasmids, despite DNA sequence similarity, are a highly diverse group of replicons.
Replicon
Rolling circle replication
Autonomously replicating sequence
Replication
Pre-replication complex
Minichromosome maintenance
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Replicon
Autonomously replicating sequence
Replication
Origin recognition complex
Replication factor C
Trans-acting
T-DNA Binary system
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Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins.
Cloning of specific regions of plasmid RSF1010, in conjunction with in vitro replication studies, has revealed three novel genes: repA, repB, and repC. They are clustered in one region of the plasmid, separated from the origin of replication by regions that are not essential for plasmid viability in an Escherichia coli host. In vivo, a 2.1-kilobase segment of the plasmid, bearing the replication origin, can establish itself as an autonomous replicon if the DNA region carrying the three rep genes is present in the same cell on an independent plasmid. In vitro, RSF1010 DNA is efficiently replicated by an ammonium sulfate fraction from the E. coli extract, provided the extracts are prepared from cells that can supply the required rep gene products. Using cells containing the cloned rep gene region as a source of elevated levels of the rep proteins, we have partially purified these proteins in functional form. When added to an enzyme fraction derived from plasmid-free cells, they specifically promote the replication of plasmid DNA bearing the RSF1010 origin.
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Replicon
Autonomously replicating sequence
Shuttle vector
Replication factor C
Ter protein
Origin recognition complex
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We have isolated a circular form of Tn2350, an IS1-flanked kanamycin resistance transposon forming part of the plasmid R1drd-19. This circle (pTn2350::9.6 kilobases) contains a single IS1 element and probably arises by recombination between the two directly repeated Is1 sequences of Tn2350. It can be used to transform Escherichia coli to kanamycin resistance. It is capable of autonomous replication but is not maintained stably in dividing cells and segregates under nonselective conditions. Cloning of a segment of pTn2350 on a conditional plasmid vector allowed us to assign the replication functions of this plasmid to a 1.6-kilobase restriction fragment. The plasmid R1drd-19 can thus be considered as a cointegrate between two replicons separated by IS1 sequences.
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Kanamycin
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Replicon
ColE1
T-DNA Binary system
Autonomously replicating sequence
Minichromosome maintenance
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