The gap between iscR and iscS was 78 bp, and insertion site of mutant
iscS + 30 was located at 48 bp downstream of iscR and 30 bp upstream of iscS. In Fe-S cluster assembly pathway, IscS is a cysteine desulfurase that procures the sulfur from cysteine for Fe-S cluster assembly ; IscR is an iron-sulphur (Fe-S) cluster containing transcription factor that represses transcription of the isc operon in E. coli, but iscRSUA operon was induced under oxidative stress [28,29]. In other bacteria, IscR was shown to both behave an activator or a repressor. Figure 7 Se(IV) resistance and reduction using different Se(IV) concentrations using four iscR insertional PF-01367338 mutants in C. testosteroni S44. The different sites of transposon insertions in iscR is given in nt from the translational start codon; +30 is an insertion upstream of iscS (A); the selleck kinase inhibitor predicted domains selleckchem of the IscR protein (B) and growth in LB medium amended with different concentrations of Se(IV) at different time points (C). The four arrows indicate the four mutants of iscR-280 (a), iscR-327 (b), iscR-513 (c) and iscS + 30 (d), respectively in A and B. The order of the 5 PA bottles of C is wild type (WT), iscR-280 (a), iscR-327 (b), iscR-513 (c) and iscS + 30 (d), respectively. The insertional mutants were more sensitive to high concentrations of Se(IV) than C.
testosteroni S44 and also grew more slowly in 10 mM Se(IV) than wild type C. testosteroni S44 . Se(IV) reduction of iscR-513 and iscS + 30 was also delayed but not as much as iscR-280 and iscR-327 (Figure 7C). The growth of iscR-280 and iscR-327 was completely inhibited in P-type ATPase 50 mM Se(IV), whereas C. testosteroni S44, iscR-513 and iscS + 30 showed
slow growth and decreased Se(IV) reduction. Those results indicated that iscR-327 was the most sensitive mutant to higher concentrations of Se(IV), followed by iscR-280 with intermediate sensitivity in iscR-513 and iscS + 30, and the highest resistance in wild type C. testosteroni S44. Despite of different resistance between wild type and iscR mutants, the presence of IscR was not essential for Se(IV) reduction. For example, in 10 mM Se(IV), iscR-280 and iscR-327 grew slowly with little apparent Se(IV) reduction and showed faint red color after 12 and 16 h incubation; in contrast, the red color due to selenium nanoparticles became similar to the wild type after 24 h incubation, indicating IscR was necessary for the growth and resistance but was not necessary for Se(IV) reduction to occur. In order to understand whether IscR influenced resistance to other heavy or transition metal(loid)s, we determined the growth of iscR mutants and the wild type. The wild type C. testosteroni S44 grew better than three iscR mutants iscR-280, iscR-327 and iscR-513 under heavy metal(loid)s such as As (III), Cu (II) and Cd (II) (Figure 8).