Article

Novel broad-host-range vehicles for cloning and shuffling of gene cassettes.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawinskiego 5A, Poland.
Journal of microbiological methods (Impact Factor: 2.43). 10/2011; 88(1):53-62. DOI: 10.1016/j.mimet.2011.10.011
Source: PubMed

ABSTRACT Novel vectors for cloning and shuffling of gene cassettes based on minireplicon of broad-host-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 (4.5-5.4kb) and carry various resistance determinants: kanamycin (Km(R)), tetracycline (Tc(R)) or chloramphenicol (Cm(R)). 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.

0 Bookmarks
 · 
76 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The broad-host-range conjugative RA3 plasmid from IncU incompatibility group has been isolated from the fish pathogen Aeromonas hydrophila. DNA sequencing has revealed a mosaic modular structure of RA3 with the stabilization module showing some similarity to IncP-1 genes and the conjugative transfer module highly similar to that from PromA plasmids. The integrity of the mosaic plasmid genome seems to be specified by its regulatory network. In this paper the transcriptional regulator KorC was analyzed. KorCRA3 (98 amino acids) is encoded in the stabilization region and represses four strong promoters by binding to a conserved palindrome sequence, designated OC on the basis of homology to the KorC operator sequences in IncP-1 plasmids. Two of the KorCRA3-regulated promoters precede the first two cistrons in the stabilization module, one fires towards replication module, remaining one controls a tricistronic operon, whose products are involved in the conjugative transfer process. Despite the similarity between the binding sites in IncU and IncP-1 plasmids, no cross-reactivity between their KorC proteins has been detected. KorC emerges as a global regulator of RA3, coordinating all its backbone functions: replication, stable maintenance and conjugative transfer.
    Plasmid 04/2013; · 1.76 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background The 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.Conclusion The MobC is an auxiliary transfer protein of dual function. It autoregulates the expression of mobC-nic operon while its presence significantly stimulates transfer efficiency.
    BMC Microbiology 09/2014; 14(1):235. · 2.98 Impact Factor

Full-text (3 Sources)

View
20 Downloads
Available from
Jun 2, 2014