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Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

ABSTRACT: IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

SUBMITTER: Kulinska A

PROVIDER: S-EPMC2446526 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

Kulinska Anna A Czeredys Magdalena M Hayes Finbarr F Jagura-Burdzy Grazyna G

Applied and environmental microbiology 20080523 13

IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and ga ...[more]

PMID: 18502921

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Project description:The broad-host-range (BHR) conjugative plasmids have developed diverse adaptive mechanisms defining the range of their promiscuity. The BHR conjugative RA3 plasmid, the archetype of the IncU group, can transfer between, replicate in, and be maintained in representatives of Alpha-, Beta-, and Gammaproteobacteria Its stability module encompasses ten open reading frames (ORFs) apparently organized into five operons, all transcribed in the same direction from several strong promoters that are tightly regulated either by autorepressors or by global plasmid-encoded regulators. In this paper, we demonstrate that owing to an efficient RNA polymerase (RNAP) read-through, the transcription from the first promoter, orf02p, may continue through the whole module. Moreover, an analysis of mRNA produced from the wild-type (WT) stability module and its deletion variants deprived of particular internal transcription initiation sites reveals that in fact each operon may be transcribed from any upstream promoter, giving rise to multicistronic transcripts of variable length and creating an additional level of gene expression control by transcript dosage adjustment. The gene expression patterns differ among various hosts, indicating that promoter recognition, regulation, and the RNAP read-through mechanisms are modulated in a species-specific manner.IMPORTANCE The efficiently disseminating conjugative or mobilizable BHR plasmids play key roles in the horizontal spread of genetic information between closely related and phylogenetically distant species, which can be harmful from the medical, veterinary, or industrial point of view. Understanding the mechanisms determining the plasmid's ability to function in diverse hosts is essential to help limit the spread of undesirable plasmid-encoded traits, e.g., antibiotic resistance. The range of a plasmid's promiscuity depends on the adaptations of its transfer, replication, and stability functions to the various hosts. IncU plasmids, with the archetype plasmid RA3, are considered to constitute a reservoir of antibiotic resistance genes in aquatic environments; however, the molecular mechanisms determining their adaptability to a broad range of hosts are rather poorly characterized. Here, we present the transcriptional organization of the stability module and show that the gene transcript dosage effect is an important determinant of the stable maintenance of RA3 in different hosts.

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

Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

Publications

Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

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