Figure 3

Analysis of mpt A regulation by FurB by heterologous expression in M. smegmatis ∆ fur B (MSMEG∆ fur B). (A) FurB amino acid sequences of MAP, Mtb and M. smegmatis (MSMEG) were compared using ClustalOmega multiple sequence alignment. Asterices indicate homologue amino acids, grey arrows show highly conserved functional sites, black arrows structural sites (according to [39]). (B) MSMEG∆furB was transformed with pMP1102, grown in MB-complete to an OD₆₀₀ of 1.0 and gene expression of mptA compared to MSMEG wildtype (wt) was analysed by qRT-PCR. Shown are the results of three independent experiments expressed as the relative fold change of gene expression of the ∆furB mutant to the wildtype, normalized to the housekeeping gene gap. Statistical analysis was performed using Mann–Whitney test with **p < 0.005. (C) MSMEG∆furB was transformed with pJEM15 or pJEM-mptA2, grown in MB-complete to an OD₆₀₀ of 1.0 treated with 10 μM TPEN for 2 h, proteins were extracted, concentration was determined and promoter activity of TPEN treated (black bars) and untreated cultures (white bars) was analysed by β-galactosidase assay. Results of at least three independent experiments (mean ± SEM) are shown. Activity was measured at a wavelength of 405 nm and related to mg protein per ml. Statistical analysis was performed by using the Kruskal-Wallis-Test with ***p >0.0001.