While there were no significant differences in β-galactosidase activity between cells grown at various temperatures (37°C and 42°C) (Figure 2A) or between cells grown in solid and liquid selleck chemical medium (MH broth and MH solidified by agar addition) (data not shown), transcription from each of the analyzed promoters was iron-regulated (Figure
2B). For cells grown in iron-restricted conditions, P dsbA2dsbBastA activity was 10 times lower, P dsbA1 activity was about 30% lower, and P dbadsbI activity was four times higher, compared to cells grown under iron-sufficient/iron-rich conditions. Figure 2 Transcription levels of C. jejuni 81-76 dsb genes Doramapimod (measured by β-galactosidase activity assays) in the wild
type strain (A and B) and fur::cat mutant (C) under different environmental conditions. Each experiment was repeated three times, and each time three independent samples were taken for each strain (giving 9 independent measurements www.selleckchem.com/products/kpt-330.html for each strain). Statistical significance was calculated using t-Student test for comparison of independent groups (GraphPad Prism). The wild type strain C. jejuni 480 carrying an empty vector pMW10 was used as a control. Statistical p values: For wild type C. jejuni 480 strain: P dba-dsbI temp. 37°C vs 42°C: p = 0,0001(*). P dsbA2-dsbB-astA temp. 37°C vs 42°C: p = 0,2020. P dsbA1 temp. 37°C vs 42°C: p = 0,1031. P dba-dsbI MH+Fe vs MH: p = 0,0576. P dba-dsbI MH-Fe vs MH: p < 0,0001(*). P dsbA1-dsbB-astA MH+Fe vs MH: p = 0,0007(*). P dsbA1-dsbB-astA MH-Fe vs MH: p < 0,0001(*). P dsbA1 MH+Fe vs MH: p = 0,2569. P dsbA1 MH-Fe vs MH: p < 0,0001(*). For mutant C. jejuni 480 fur::cat strain: P dba-dsbI
MH+Fe vs MH: p = 0,3683. P dba-dsbI MH-Fe vs MH: p = 0,6796. P dsbA1-dsbB-astA MH+Fe vs MH: p = 0,3164. P dsbA1-dsbB-astA MH-Fe vs MH: p = 0,0577. P dsbA1 MH+Fe vs MH: p = 0,5228. P dsbA1 MH-Fe vs MH: p = 0,2388. P values of P < 0.05 were considered to be statistically significant; they are marked with (*). Iron-regulated expression of many Gram-negative bacterial genes is mediated by the ferric uptake regulator (Fur) [35, 36]. Classically, the Fur protein first binds to its co-repressor Fe2+ , and then binds to the conserved Phospholipase D1 DNA sequence (Fur-box) of the regulated promoter, repressing its transcription. However, transcriptomic analyses documented that apo-Fur (without complexed co-repressor) can also influence gene transcription in response to iron concentration [6, 36–38]. We therefore decided to evaluate the regulatory function of the Fur protein on dsb gene expression. For this purpose a C. jejuni 480 fur isogenic mutant was constructed. Then, recombinant plasmids containing dsb promoter-lacZ fussions (pUWM803, pUWM864 and pUWM827) were introduced into the C. jejuni 480 fur::cat mutant by electroporation.