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.

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.