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.

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.

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.

No abstract

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 ...