With this fusion protein, we established a directed transposon mu

With this fusion protein, we established a directed transposon mutagenesis system that is expected selleck products to directly integrate close to the fliC operator. The system is composed of three main elements: (1) the target fliC operator flanked by fliC and fliD genes; (2) the IS30–FljA fusion transposase;

(3) and the integration donor sequence containing the (IS30)2 intermediate together with the CmR marker gene. Two essential components of the mutagenesis system required to be constructed: the fusion transposase producer plasmid pFOL1111 and the integration donor pFOL1069 (Fig. 2). The insertion donor plasmid pFOL1069 containing the (IS30)2 intermediate (Olasz et al., 1993; Kiss & Olasz, 1999) represented a highly reactive DNA segment in the presence of the IS30 transposase. The pFOL1069 additionally contained

the CmR marker gene, the mob region necessary for Ku-0059436 purchase bacterial conjugation and the defective replication origin R6K. Because of the R6K replication origin, the donor plasmid is unable to replicate in Salmonella lacking the pir gene. As a consequence, Salmonella bacteria possessing CmR after the conjugation of pFOL1069 were the ones in which the donor plasmid was integrated into the chromosome. The integration ability of pFOL1069 was verified earlier in E. coli (data not shown). The FljA–IS30 fusion transposase producer plasmid pFOL1111 was constructed by the fusion of the fljA flagellin repressor gene to the C-terminal end of the IS30 transposase gene. The resulting isopropyl-β-d-thiogalactopyranoside-inducible FljA-transposase producer plasmid

also contains the ApR bacterial marker. Because this plasmid codes only for the fusion transposase, but lacking the IS30 inverted repeat ends necessary for transposition, it is not capable of integrating into any target DNA. The inducible expression of the FljA–IS30 fusion protein was verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (Fig. 3a). No alteration was detected in the amount of the transposase compared with that of the wild type produced by the plasmid pJKI132 (Fig. 3a). The functionality of selleck chemicals the FljA part of the fusion was tested by introducing pFOL1111 into the wt S. Enteritidis strain 11 and the motility of the transformants was investigated. The pFOL1111 plasmid-harbouring strains (Fig 3b, column 2) showed reduced motility as compared with the plasmid-free bacteria (Fig 3b, column 1). However, a complete elimination of motility never occurred due to the presence of exogenous FljA, and it was always reversible, as the partly motile strains regained their full motility after the plasmid pFOL1111 was eliminated (results not shown). The transposition activity of pFOL1111 was verified similarly as described by Szabo et al. (2008) (data not shown). In summary, it can be stated that all components of the targeting system have proven their expected activity for subsequent immobilization.

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