Figure 5.

Creation of new IPTG and arabinose-inducible expression systems in C. burnetii. (A) Schematics of two different IPTG-induction systems allowing inducible expression of mCherry and their respective immunoblots detecting mCherry production. The upper schematic shows the original IPTG-inducible system contained on the pJB-CAT and pKM230 C. burnetii shuttle vectors, which consists of a truncated lacI gene (missing the tetramerization domain), a tac promoter, and a single LacI binding site upstream of mCherry. Production of mCherry was seen in the minus IPTG induction by immunoblot (upper right) indicating leaky expression of mCherry. The lower schematic shows the new IPTG-inducible system consisting of a full-length lacI gene and lacO promoter containing dual LacI binding sites upstream of mCherry. No background mCherry production was seen in the uninduced control, whereas good production of mCherry was evident in the IPTG-induced sample. (B) Schematic of the arabinose-induction system allowing inducible expression of mCherry. The mCherry gene was cloned downstream of the araB promoter. The sequence of the native araB promoter is shown. This sequence was modified so that the -35 and -10 regions had a smaller 16-bp spacer region and their sequences were closer to the consensus sigma 70 promoter sequence (TTGACA and TATAAT, respectively). The native and modified arabinose induction systems were cloned into the pJB-CAT shuttle vector and then transformed into C. burnetii. Arabinose-based gene expression was examined by immunoblot of 0-, 4-, or 7-h post-arabinose induction of 5-day C. burnetii cultures. Production of mCherry from the native araB promoter was very low at 7 h, whereas in the modified araB system, high levels of protein were detected at both 4 and 7 h post induction. No background mCherry production was seen in the uninduced control (0 h).