Metal dependent regulation of a M. avium ssp. paratuberculosis specific gene locus. MAPwt was grown in MB-complete to an OD600 of 1.0 and treated with different chelating agents and supplements as described in Methods. After RNA extraction, changes in gene expression levels of mbtB (black bars), mptA (white bars) and sidA (grey bars) were analysed by qRT-PCR. (A) 200 μM 2,2-bipyridyl (DIP) for 2 h. (B) 14 mM nitrilotriacetic acid (NTA) for 24 h. (C) NTA treated cultures (14 mM, 24 h) supplemented with 1 mM ZnSO4, FeSO4, MgCl2, CaCl2, CuSO4, CoCl2 or MnSO4. (D) 10 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) for 2 h. (E) TPEN treated cultures (10 μM, 2 h) supplemented with ZnSO4 or FeSO4 both in a final concentration of 7.5 μM. Shown are the results of at least three independent experiments (mean ± SEM). Results were normalized to the housekeeping gene gap and are expressed as fold change compared to the untreated controls. Statistical analyses were performed using Kruskal-Wallis test (C) with *p < 0.01 and ***p < 0.0001 or Mann–Whitney test (E) with ***p < 0.0001.