coli control strains, ruling out the possibility that the probe shift was due to non-specific binding of contaminating proteins. A comparable shift was observed for recombinant SO2426sh (Figure 6B), thus supporting our proposition that the
actual 5′ terminus of the SO2425 occurs at residue M11. Gel shift assays performed with additional DNA probes upstream of the so3030-3031-3032 operon as well as so3036, which also contains a putative SO2426 recognition sequence, showed a band-shift in the presence of recombinant SO2426 (data not shown). Although the primary focus in this study is the functional role of SO2426 in siderophore selleck chemicals production, future studies will be necessary to analyze the interaction of SO2426 with additional recognition sites to further define CBL0137 chemical structure its regulon. Figure 5 Upstream nucleotide sequence of the siderophore biosynthesis so3030 – 3031 – 3032 operon. The recognition site (Fur Box) for the ferric uptake regulator (single underline) and the predicted SO2426-binding motif (red type) are noted in the upstream region. A DNA probe for EMSA studies flanking the SO2426-binding motif was generated by PCR amplification
(double underlined sequence). The 5′ coding region of so3030 is highlighted in salmon. Figure 6 Binding of recombinant SO2426 proteins to putative recognition site. Electrophoretic mobility shift assays were performed to demonstrate binding of recombinant SO2426 (A) and SO2426sh (B) to the predicted SO2426 recognition motif upstream of the so3030-3031-3032 operon. Lanes: 1, DNA template only; 2, vector-only control E. coli cell lysate (15 μg); 3-7, increasing concentrations of either recombinant SO2426 or SO2426sh XAV-939 ranging from 0.6 to 3.0 μg in 0.6 μg increments. Each reaction
mixture contained 95 ng of DIG-labeled DNA template. No binding was seen with an excess of vector-only control cell lysates (lane 2); whereas, a clear shift is seen with increasing amounts of either recombinant SO2426 or SO2426sh. Siderophore production is deficient in a Δso2426 mutant strain Earlier physiological evidence for the role of SO2426 PLEKHM2 in siderophore production was obtained using liquid CAS assays in which relative siderophore production levels for the Δso2426 mutant were compared to those for the wild-type MR-1 strain [21]. These studies demonstrated that the deletion mutant was markedly deficient in siderophore synthesis compared to the wild-type strain in LB medium supplemented with chromate [21]. LB medium constitutes a sufficient source of iron (~17 μM) [13]. Additionally, under iron-replete conditions, in which 50 μm FeCl3 was added to the medium, there was no change in siderophore levels in the Δso2426 mutant. Conversely, siderophore production in the wild-type MR-1 strain returned to background levels in the presence of added iron [21].