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The broad-host-range 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 and be maintained in representatives of Alpha-, Beta- and Gammaproteobacteria. Its stability module encompasses ten 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 read-through, the transcription from the first promoter, orf02p, may continue through the whole module. Moreover, an analysis of mRNA produced from the 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 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 plasmids' promiscuity depends on the adaptations of its transfer, replication and stability functions to the various hosts. IncU plasmids, with the archetype 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 gene transcript dosage effect is an important determinant of the RA3 stable maintenance in different hosts.

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.

The aim of the study was to evaluate the effect of MAP and film thickness on Agaricus bisporus quality. Three initial gas compositions: high oxygen packaging (100% of O2); medium oxygen packaging (50% O2, 50% N2); low oxygen packaging (5% O2, 5% CO2, 90% N2) and two types of polyethylene films of 54 and 39 µm thickness were used. The samples were stored at 4 ± 1C for 14 days. The films and MAP had a significant effect on color and weight loss of the mushrooms. In the case of texture, the gas composition alone exerts a significant effect. The optimal conditions for prolonging the shelf life of mushrooms were found for packaging with medium oxygen level and film of higher permeability. In optimal conditions, mushrooms were characterized by lightness of 93 and 85, and a browning index of 11 and 23 in 0 and 14 days of storage, respectively. Practical Application White button mushrooms are highly perishable. The average storage time of fresh mushrooms does not exceed 4 days. There is a pressing need for effective methods to extend the shelf life of these mushrooms. According to the presented results, it is possible to maintain the product visually acceptable up to 14 days for mushrooms packed with the usage of a 39 µm thick film type and modified atmosphere with a medium level of oxygen (50%).

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 ...

Summary Conjugative transfer of the broad‐host‐range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC‐nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC‐nic operon. A 7‐bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3 was delineated to a 47‐bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM. This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.

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