Upstream of astA and dsbA1 there are putative RBS sequences and incomplete promoter nucleotide sequences, suggesting that astA and dsbA1 might be transcribed separately from dsbA2 and dsbB. Figure 1 Organization of dsb genes in the C. jejuni 81-76 chromosome and constructs prepared for dsb transcription studies; the dsbA2-dsbB-astA-dsbA1 gene set (A), the dba-dsbI gene set (B). Hazy grey boxes stand for C. jejuni genes (C. jejuni NCTC 11168 and 81-176 gene numbering is given above the boxes, below them the studied gene names are given). Black boxes stand for the C. jejuni 81-176 DNA fragments

cloned in the transcriptional fusions with the promoterless lacZ gene, displayed by the light grey boxes. The longest transcriptional fusion could not be obtained. Sign β-gal +/- at the right Akt inhibitor side of the plasmid name stands for presence/absence of β-galactosidase activity conferred

by the appropriate construct for the transformant cells. C. jejuni 81-176 dba (cjj81176_0045c) and dsbI (cjj81176_0044c) have the same orientation in the chromosome (Figure 1B) and their coding sequences are separated by a short intergenic region of 11 bp. An initial RT-PCR experiment carried out on the total C. jejuni RNA documented dba-dsbI co-transcription in vitro and localization of their promoter within 493 bp DNA upstream of see more the dba translation start codon [18]. Transcriptional analysis of two dsb gene clusters The lacZ reporter gene system was used to determine the dsb gene expression and regulation. Two sets of dsb-lacZ transcriptional fusions were designed based PAK5 on a promotorless lacZ gene in the shuttle vector pMW10 [34]. The first one comprised of seven plasmids (pUWM792, pUWM795, pUWM803, pUWM832, pUWM833, EPZ015938 supplier pUWM834 and pUWM864) employed to study dsbA2/dsbB/astA/dsbA1 expression. The other consisted of three plasmids (pUMM827, pUWM828 and pUWM858) generated to analyze dba/dsbI expression. Details of the recombinant plasmid structures are shown in Figure 1. We successfully prepared all but one of the planned transcriptional fusions – we failed at constructing

the longest fusion presented in Figure 1. β-galactosidase assays indicated that the fusions present in pUWM833, pUWM834 and pUWM858 were not expressed in C. jejuni cells. This documented that the analyzed genes form two polycistronic operons (dsbA2-dsbB-astA and dba-dsbI) and only dsbA1 is independently transcribed. The level of β-galactosidase provided by the dsbA1 promoter was approximately ten times higher than that conferred by the two other promoters that were analyzed (contained in pUWM803 and pUWM827). Thus, three promoters of various strengths and responsible for C. jejuni dsb gene expression were identified: P dbadsbI , P dsbA2dsbBastA and P dsbA1 . Influence of environmental stimuli on dsb gene expression We subsequently tested whether gene expression driven by P dsbA2dsbBastA , P dsbA1 and P dbadsbI (C.