jejuni Δdba-dsbI::cat (AG6) – was constructed. Thereafter three recombinant shuttle plasmids, pUWM769 (containing the wild type C. jejuni dba-dsbI operon), pUWM811 and pUWM812 (containing point mutated dba – M1R or dba: L29stop, respectively, and wild type dsbI) were introduced into mutant cells. Transformant cells were screened for DsbI synthesis by Western blot analysis with specific rabbit anti-rDsbI serum and additionally by RT-PCR for the presence of dsbI transcript. Introduction of pUWM769 Selonsertib ic50 into C. jejuni 81-176 AG6 (Δdba-dsbI::cat), cells resulted in restoration of DsbI production

in a higher amount compared to the wild type strain (Figure 4, lane 6), due to plasmid-encoded dba-dsbI gene expression. When dba translation was completely aborted (C. jejuni AG6 carrying pUWM811) and when the truncated 28 aa Dba was produced (C. jejuni AG6/pUWM812), DsbI was not synthesized at all (Figure 4, lane 4 and 5, respectively). RT-PCR experiments proved that point mutations in dba did not influence dsbI transcription, as comparable amounts

of dsbI mRNA were detected in all but one (AG6) of the strains (Figure 5, lanes: 9, 11-13). Comparable results were obtained for series of C. jejuni dsbI::cat strains carrying pUWM769, pUWM811 and pUWM812 Repotrectinib supplier plasmids (data not shown), suggesting that intact, chromosomally-encoded Dba cannot act in-trans to ensure dsbI mRNA translation. Figure 4 Translational coupling of C. jejuni dba – dsbI. Western blot (anti-rDsbI) analysis of C. jejuni protein extracts separated by 12% SDS-PAGE. Relative positions of molecular weight markers (lane 1) are listed on

the left (in kilodaltons). Lanes 2-6 contain 15 μg of total proteins from: C. jejuni 81-176 Glutathione peroxidase wt (2), C. jejuni 81-176 AG6 (dba-dsbI::cat) (3), AG6/pUWM811 (4), AG6/pUWM812 (5) and AG6/pUWM769 (6) Figure 5 Analysis of C. jejuni dsbI transcription from a dba-dsbI operon containing wild type or point mutated dba. RT-PCR analysis of dsbI (and aphA-3) transcription in C. jejuni wild type and mutant cells. Equal amounts of mRNAs isolated from C. jejuni cells were reverse-transcribed using primer KM-R1 or Cj-RT and resulting cDNA was Saracatinib purchase PCR-amplified with primer pairs KM-L1 – KM-R1 (lanes 1-7) or CjNde – Cj17RM (lanes 8-14), respectively. Relative positions of DNA molecular length markers (lanes 1, 8) are listed on the left (in base pairs). Lanes 2-6 and 9-13 contain PCR products amplified on cDNAs for C. jejuni 81-176 wt (2, 9), AG6 (dba-dsbI::cat) (3, 10), AG6/pUWM811 (4, 11), AG6/pUWM812 (5, 12), AG6/pUWM769 (6, 13); lanes 7 and 14 contain PCR products amplified on RNA for AG6/pUWM769 (after DNase treatment). White arrows indicate products of expected size. To further address the role of Dba in dsbI expression the recombinant plasmid lacking the dba gene but containing the dsbI gene transcribed from own promoter was constructed and introduced into the C. jejuni 81-176 Δdba-dsbI::cat mutant. The C.