8 kb cat gene excised from pRY109) was inserted in the same trans

8 kb cat gene excised from pRY109) was inserted in the same transcriptional orientation as dba-dsbI operon at the BamHI site between the C. jejuni DNA fragments, generating suicide plasmid pUWM866. Gene versions buy SP600125 inactivated by insertion of a resistance cassette were introduced into the C. jejuni 81-176 or 480 chromosome by the allele exchange method as described by Wassenaar et al. [24]. Construction of the C. jejuni 480 fur::cat mutant was achieved by natural transformation using C. jejuni 81-176 fur::cat chromosomal DNA. It should be pointed out that C. jejuni 480 was previously described as incapable of accepting chromosomal DNA by natural transformation [24]. Such inconsistency of experimental data

might be due to different chromosomal DNA used for natural transformation (C. jejuni 81116 vs C. jejuni 81-176). The mutant strains were obtained by two- or tri-parental mating experiments PND-1186 clinical trial performed as described by Labigne-Roussel et al. [29] and Davis et al. [30]. The constructed mutants were named AG1 (C. jejuni 81-176 dba::aphA-3), AL1 (C. jejuni 81-176 dsbI::cat),

AL4 (C. jejuni 480 dsbI::cat), AG6 (C. jejuni 81-176 Δdba-dsbI::cat), AG11 (C. jejuni 81-176 fur::cat), and AG15 (C. jejuni 480 fur::cat). They demonstrated normal colony morphology and all but two had normal growth rates when cultured on BA plates. Only the C. jejuni 81-176 fur::cat and C. jejuni 480 fur::cat exhibited slower KPT-8602 manufacturer growth, an observation consistent with other studies on fur mutants [25]. Disruption of each gene as a result of double cross-over recombination was verified by PCR with appropriate pairs of primers flanking the insertion site (Table 2). The loss of DsbI synthesis in the constructed mutants was verified by Western blotting of whole-cell protein extracts against specific rabbit polyclonal Calpain anti-rDsbI antibodies. Protein manipulation, and β-galactosidase and arylsulfate sulfotransferase (AstA) assays Preparation of C. jejuni protein extracts, SDS-PAGE (sodium dodecyl sulfate polyacrylamide

gel electrophoresis) and blotting procedures were performed by standard techniques [26]. To obtain recombinant His6-DsbI protein, the 1100 bp DNA fragment containing the coding sequence for the predicted periplasmic DsbI C-region was PCR-amplified from the C. jejuni 81-176 chromosome using a primer pair: Cj17WDBam-up – Cj17WDBam-low. This fragment was cloned into the pGEM-T Easy vector and then, using BamHI restriction enzyme, into expression vector pET28a (Novagen) to generate plasmid pUWM657, whose correct construction was verified by restriction analysis and sequencing. Cytoplasm-located soluble fusion protein His6-DsbI purified from the E. coli Rosetta (DE3) LacIq strain by affinity chromatography was used for rabbit immunization (Institute of Experimental and Clinical Medicine, Polish Academy of Science, Warsaw, Poland).

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