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.Conjugative plasmids contribute greatly to the global spread of genetic information and gene exchange, as in some cases they can self-transfer even between distant bacterial species. Conjugative R factors assigned to the IncU incompatibility group have been isolated from a number of Aeromonas spp. and Escherichia coli strains from seawater fish hatcheries and diseased fish, as well as from clinical environments (2,4,6,15,26,30,31,42). Members of the IncU plasmid group are implicated particularly in the dissemination of antibiotic resistance in Aeromonas strains associated with aquatic environments (15).IncU representatives pAr-32 and pRAS1 contain resistance genes encoded within integrons, and on the basis of restriction enzyme analysis of both plasmid molecules, Sørum et al. (35) postulated a highly conserved backbone structure of IncU group members with variability in the region coding for antibiotic resistance. Similar observations were made with plasmids pASOT and pFBAOT (2,26,27). Plasmid pFBAOT6 (84,749 bp) has been sequenced recently and analyzed in silico (27). Plasmid Rms149 of the Pseudomonas IncP-6 group was assigned to the IncU group on the basis of incompatibility tests (12). Apart from homology between the replication genes of pFBAOT6 and Rms149, no conservati...

RA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative ؊35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over ϳ300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The ϳ300-bp fragment also encompasses the origin of conjugative transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.Plasmids from the IncU incompatibility group have been isolated from the fish pathogens Aeromonas salmonicida and Aeromonas hydrophila in worldwide locations (4,5,6,12,19,20,44), as well as from clinical isolates of Escherichia coli and A. hydrophila (40,53). The archetypal IncU plasmid is the low-copy-number, conjugative RA3 plasmid (45,909 bp) isolated from A. hydrophila in Japan in 1979 (5), which confers resistance to sulfonamides, streptomycin, and chloramphenicol. RA3 transfers with high frequency from E. coli to Pseudomonas putida (Gammaproteobacteria) and Ralstonia eutropha (Betaproteobacteria) and with three-magnitude-lower efficiency between E. coli and Agrobacterium tumefaciens (Alphaproteobacteria). RA3 is stably maintained in all tested representatives of these three classes (31). The nucleotide sequence of RA3 (GenBank accession no. DQ401103) (31) revealed the existence of putative functional modules involved in replication, maintenance, and conjugative transfer that are almost identical to those of the tetracycline-resistant IncU plasmid pFBAOT6 (41). The sequence confirms a high degree of conservation in the backbone functions of IncU plasmids suggested previously, on the basis of restriction enzyme analysis (47). Particularly interesting features of the RA3/pFBAOT6 backbone include a replication module with similarity to several unrelated environmental plasmids from a wide diversity of beta-and gamm...

Broad-host-range conjugative RA3 plasmid of IncU incompatibility group has been isolated from fish pathogen Aeromonas hydrophila. DNA sequencing revealed mosaic modular structure of RA3 with stabilization module showing some degree of similarity to IncP-1 genes whereas conjugative transfer module being highly similar to PromA plasmids. The integrity of mosaic plasmid genome seems to be specified by its regulatory network. In this paper the transcriptional regulator KorC has been analyzed. The KorCRA3 (98 amino acids) is encoded in the stabilization region and it represses five strong promoters by binding to the conserved palindrome sequence, designated OC on the basis of homology to KorC operator sequences in IncP-1 plasmids. Two of KorCRA3 regulated promoters precede the first two cistrons in the stabilization module, and one fires towards replication module. Among two other divergently oriented back-to-back promoters, one is upstream of the long transcriptional unit of 19 orfs, products of which are predicted to be involved in the conjugative transfer process and another controls tricistronic operon encoding proteins of unknown functions. Despite the similarity between binding sites in IncU and IncP-1 plasmids no cross-reactivity between KorC proteins has been detected. The KorC emerges as the global regulator in RA3 coordinating all plasmid backbone functions: replication, stable maintenance and conjugative transfer.

BackgroundThe IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups. Despite its frequent occurrence the mechanisms of relaxosome formation/action are far from understood. Here we analyzed the putative transfer auxiliary protein MobC of the conjugative plasmid RA3 from the IncU incompatibility group.ResultsMobC is a protein of 176 amino acids encoded in the bicistronic operon mobC-nic adjacent to oriT. MobC is homologous to prokaryotic transcription factors of the ribbon-helix-helix (RHH) superfamily. Conserved LxxugxNlNQiaxxLn motif clusters MobC with the clade of conjugative transfer auxilliary proteins of MobP relaxases. MobC forms dimers in solution and autoregulates the expression of mobCp by binding to an imperfect palindromic sequence (OM) located between putative -35 and -10 motifs of the promoter. Medium-copy number test plasmid containing the oriT-mobCp region is mobilized with a high frequency by the RA3 conjugative system. The mutations introduced into OM that abolished MobC binding in vitro decreased 2-3 fold the frequency of mobilization of the test plasmids. The deletion of OM within the RA3 conjugative module had no effect on transfer if the mobC-nic operon was expressed from the heterologous promoter. If only nic was expressed from the heterologous promoter (no mobC) the conjugative transfer frequency of such plasmid was 1000-fold lower.ConclusionThe MobC is an auxiliary transfer protein of dual function. It autoregulates the expression of mobC-nic operon while its presence significantly stimulates transfer efficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0235-1) contains supplementary material, which is available to authorized users.

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The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parS RA3 , forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nic operon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding Large, low-copy-number plasmids displaying a broad host range (BHR) carry an extended backbone of operons involved in replication, copy number control, maintenance, and in the case of self-transmissible plasmids, also conjugative transfer. Expression of these genetic units is driven by high-activity promoters and therefore imposes a substantial metabolic cost on the bacterial cell. To minimize the metabolic burden to the host, regulatory networks have evolved to diminish the backbone gene expression to a low basal level while still allowing for rapid upregulation of the transcription when needed. The regulation is achieved by autogenous repressors that produce negative-feedback loops, mediumrange repressors that control particular modules, and globally acting regulators that bind operators scattered along the plasmid molecule to coordinate expression of all modules. Fine-tuning of gene expression is postulated to be achieved by the action of corepressors and their cooperative binding in the promoter regions, which makes the plasmid highly responsive and facilitates its adaptation (1-4).BHR plasmids from the incompatibility group IncU are widespread and ubiquitous in various aquatic environments, freshwater, fish farms, and clinical isolates (5-8). The modular-mosaic backbone of the IncU plasmids is extremely well conserved not only in its overall genetic organization but also at the nucleotide sequence level (8-10). The mosaic character of genomes from this group is demonstrated by homology of the functional blocks involved in replication, stable maintenance, and conjugative transfer to the respective functional modules of plasmids from different incompatibility groups. Thus, the stability region of RA3 encodes seven homologs of IncP-1 proteins (11), whereas the RA3 conjugative transfer region clusters with similarly organized modules from the PromA group of plasmids (12-16). The RA3 plasmid (Fig. 1), the group archetype isolated from the aquatic bacterium Aeromonas hydrophila (17), is the best-studied IncU representative. Its DNA sequence has been established (GenBank accession no. DQ401103) (10), and individual functional modules have been analyzed ...

Novel vectors for cloning and shuffling of gene cassettes based on minireplicon of broadhost-range RA3 plasmid from IncU incompatibility group were constructed. A series of minireplicon variants were prepared with copy number ranging from low (1-2 copies per chromosome), medium (10-15 copies per chromosome) to high copy number (80-90 copies per chromosome). The new cloning vectors are relatively small in size (4.5-5.4 kb) and carry various resistance determinants: kanamycin (KmR), tetracycline (TcR) or chloramphenicol (CmR). The vectors were engineered to facilitate cloning and shuffling of the functional modules with or without transcriptional terminators. Using the described strategy, a bank of functional modules, ready for exchange, has been initiated. Szarlak, Anna Kulińska and Grażyna Jagura-Burdzy. We present the novel broad-host-range vectors based on minireplicon of RA3 plasmid from IncU incompatibility group. Described plasmids were constructed for easy cloning and functional analysis of gene cassettes in the various bacterial species. They also offer the opportunity of gene cassettes shuffling to create "synthetic" plasmids. I am hoping you will consider the manuscript appropriate for publication in Journal of Microbiological Methods. To Yours sincerelyGrazyna Jagura-Burdzy Cover LetterMinireplicon of RA3 plasmid from IncU incompatibility group was exploited to create novel vectors for cloning and gene cassette shuffling in bacteria. New vectors are capable of replication in a broad range of hosts, are relatively small in size and carry various resistance determinants. The low, medium and high copy number vector variants were constructed. The vectors were engineered to facilitate cloning and shuffling of the functional modules with or without transcriptional terminators. Using described system gene cassettes may be easily linked together and exchanged creating novel plasmids with desired properties for various applications. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 *Highlights AbstractNovel vectors for cloning and shuffling of gene cassettes based on minireplicon of broadhost-range RA3 plasmid from IncU incompatibility group were constructed.

BackgroundLow-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use.ResultsTo facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ reporter systems facilitate easy monitoring of plasmid segregation.ConclusionThe range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species.