Functional characterization and metal ion specificity of the metal-citrate complex transporter from Streptomyces coelicolor

Secondary transporters of citrate in complex with metal ions belong to the bacterial CitMHS family, about which little is known. The transport of metal-citrate complexes in Streptomyces coelicolor has been investigated. The best cofactor for citrate uptake in Streptomyces coelicolor is [Fe.sup.3+], but uptake was also noted for [Ca.sup.2+], [Pb.sup.2+], [Ba.sup.2+], and [Mn.sup.2+]. Uptake was not observed with the [Mg.sup.2+], [Ni.sup.2+], or [Co.sup.2+] cofactor. The transportation of iron-and calcium-citrate makes these systems unique among the CitMHS family members reported to date. No complementary uptake akin to that observed for the CitH ([Ca.sup.2+], Ba2+, Sr2+) and CitM ([Mg.sup.2+], [Ni.sup.2+], [Mn.sup.2+], [Co.sup.2+], [Zn.sup.2+]) systems of Bacillus subtilis was noted. Competitive experiments using EGTA confirmed that metal-citrate complex formation promoted citrate uptake. Uptake of free citrate was not observed. The open reading frame postulated as being responsible for the metal-citrate transport observed in Streptomyces coelicolor was cloned and overexpressed in Escherichia coli strains with the primary [Fe.sup.3+]-citrate transport system OrecABCDE) removed. Functional expression was successful, with uptake of [Ca.sup.2+]-citrate, [Fe.sup.3+]-citrate, and [Pb.sup.2+]-citrate observed. No free-citrate transport was observed in IPTG (isopropyl-13-D-thiogalactopyranoside)induced or -uninduced E. coli. Metabolism of the [Fe.sup.3+]-citrate and [Ca.sup.2+]-citrate complexes, but not the [Pb.sup.2]+-citrate complex, was observed. Rationalization is based on the difference in metal-complex coordination upon binding of the metal by citrate.