Plasmid pCU1 Research Articles

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.

Mutations within the replicon of the IncN plasmid pCU1 that affect its Escherichia coli polA-independence but not its autonomous replication ability

The minimal replicon of the incompatibility N group plasmid pCU1 is contained within a 2-kb DNA region of the plasmid. The ability of this region and of the deletion derivatives thereof, that are capable of autonomous maintenance, to direct polypeptide synthesis was examined. Two proteins of 27 and 5.5 kDa are encoded by the minimal replicon. Polypeptide chain-terminating mutations within the predicted open reading frame for the 27-kDa polypeptide abolished the synthesis of this polypeptide and also the Escherichia coli polA-independence phenotype of the pCU1 replicon. However, these mutations did not affect the autonomous replication ability of the pCU1 replicon in wild-type E. coli and the expression of incompatibility towards the parental plasmid.

Host Ranges of the IncN Group Plasmid pCU1 and Its Minireplicon in Gram-Negative Purple Bacteria

The bacterial host ranges of the conjugatively self-transmissible IncN group plasmid pCU1 and its mobilizable miniderivative, pCU785, were examined. Species of the gram-negative purple bacteria were chosen for this study. Conjugative mobilization of pCU785 into a wide variety of bacteria was facilitated by the presence of oriT of the broad-host-range plasmid RK2 in pCU785. Although the host range of the IncN tra system of pCU1 is broad, the host range of its replicon is limited. However, the pCU1 replicon can be maintained in Agrobacterium, Bradyrhizobium, and Rhizobium species under conditions that select for plasmid maintenance. It is lost efficiently from these populations on release of selection.

Isolation and structure of the replicon of the promiscuous plasmid pCU1

Evidence is presented to indicate that a PvuII fragment of approx. 2 kb isolated from the 39-kb IncN-group plasmid pCU1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12. The fragment was sequenced. The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group. There are three open reading frames within the fragment. We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features.

Regions on plasmid pCU1 required for the killing of Klebsiella pneumoniae.

Plasmid pCU1 was Kik+ (promotes killing of Klebsiella pneumoniae). All Tn5 insertions within the tra region of pCU1 were Kik-. Two other regions, kikA and kikB, were needed. They may be separated on different plasmids, but both must be mobilized into Klebsiella pneumoniae. Establishment of one kik region in K. pneumoniae followed by receipt of the second did not lead to killing. Kik was therefore intracellular and required concerted and transient action of both regions.

N conjugative transfer system of plasmid pCU1.

The mating and plasmid DNA transfer functions in Escherichia coli K-12 strains that are determined by the IncN group plasmid pCU1 are specified by a single 19.4-kilobase (kb) segment of the 39-kb plasmid. Analysis of this tra region by transposon Tn5 mutagenesis and by complementation tests indicated that a subset of tra comprising six complementation groups (presumably transcription units) determines sensitivity to the N pilus-specific bacteriophages IKe and PRD1. Deletion derivatives and molecular clones that contained only this part of tra conferred sensitivity to IKe and PRD1 and the ability to produce N pili without conferring the Tra phenotype. The entire tra region contained two other complementation groups. If the 19.4-kb tra region is interrupted by DNA that is irrelevant to the Tra phenotype, such DNA must be less than 2 kb long. There are two regions within tra where this is a possibility. These observations are compared with those obtained recently with a related plasmid.

Inverted repeats in the DNA of plasmid pCU1.

Renaturable regions in the DNA strands of the N group plasmid pCU1 have been visualized as stem-loop structures by electron microscopy. Four such distinct structures are described, the smallest of which is within the loop of a larger one. The region of pCU1 in which these structures occur has several restriction sites. This and the availability of plasmid deletions and recombinants has permitted the mapping of these structures relative to one another and to the restriction and functional map of the plasmid. The replication and maintenance region of the plasmid is located within one of these stem-loop structures.

Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli

HindIII restriction sites were created artificially by the insertion of the transposon Tn.5 into the IncN plasmid pCU1 near a presumptive end of its conjugal transfer region ( tra). This allowed cloning of an entire and continuous 19.4-kb region of this plasmid that specifies the N transfer system. The cloning vector was the nonconjugative plasmid pACYC184. The recombinant plasmid was as efficient in transfer as the parental N plasmid. Other clones and deletions extending into the tra region allowed localization of a 11.2-kb segment of this region that determines sensitivity to the N-specific bacteriophages IKe and PRD1. It could also be concluded that the ability of pCU1 to promote the killing of Klebsiella pneumoniae requires a 2-kb region that is not part of, but adjacent to the tra region.

Physical and genetic organization of the IncN-group plasmid pCU1

A restriction endonuclease-cleavage map of the IncN group plasmid pCU1 was constructed. Deletion mutants of the plasmid were obtained by in vivo or in vitro methods. Comparison of the restriction maps of these mutants to that of pCU1 enables one to assign the known functions of the plasmid to particular regions on the plasmid DNA. For different enzymes, the number and distribution of restriction sites on pCU1 is compared to that of other IncN and related plasmids.