Functional Characterization and Metal Ion Specificity of the Metal-Citrate Complex Transporter from Streptomyces coelicolor

Citrate is a primary metabolite that is ubiquitously used as a source of carbon and energy by most living organisms. While there has been extensive research conducted on citrate transport across membranes (2), there has been a relative dearth of research on membrane protein systems that can transport complexed citrate (21). Currently, there is predicted to be over 90 members in the so-called CitMHS family of secondary transporters, according to the UC San Diego Transport Classification Database (http://www.tcdb.org). Members of this superfamily are found in gram-positive bacteria and are predicted in gram-negative bacteria. It is believed that these organisms take up complexed citrate, because it is predominantly available as such in their environment, or they allow access to critical metal ions, such as iron. To date, the only functionally characterized systems for metal-citrate transport in this family are those of Bacillus subtilis (21), Streptococcus mutans (20), and, most recently, Enterococcus faecalis (5). These members of the CitMHS family transport metal-citrate complexes in symport with one H+ ion per M2+ (metal)-citrate complex. Krom et al. demonstrated that CitM from B. subtilis transported citrate in complex with Mg2+, Ni2+, Mn2+, Co2+, and Zn2+ but not in complex with Ca2+, Ba2+, or Sr2+ (21). CitH, also from B. subtilis, transported citrate and isocitrate complexed with Ca2+, Ba2+, and Sr2+ but not with Mg2+, Ni2+, Mn2+, Co2+, or Zn2+ (28). Clearly, the group of metal ions transported by CitM includes the smaller cations, with a Pauling radius of less than ∼0.80 A. The ions transported by CitH of B. subtilis have radii larger than 0.98 A. Neither transporter was shown to transport free citrate or metal complexes of other tricarboxylates (or similar dicarboxylates), such as cis-aconitate and tricarballylate. More recently, Korithoski et al. functionally characterized the CitM homolog from S. mutans (20). Citrate complexed with Fe3+ and Mn2+ was transported with S. mutans, but citrate complexed with Ca2+, Mg2+, and Ni2+ was not. Korithoski et al., in fact, state that iron is the most efficient cofactor for citrate uptake in S. mutans (20). This suggests the intriguing possibility, given that S. mutans is considered the major etiological agent of dental cavities, that the organism may use the system to access essential iron, and so the system may play a role in pathogenesis. Because members of the CitMHS family are postulated to occur in bacteria such as Bacillus anthracis, Mycobacterium tuberculosis, and Corynebacterium diphtheriae, further dimension is added to our desire to understand these systems and their possible contribution to pathogenicity. The CitH transporter of Enterococcus faecalis was characterized in 2006 (5). High amino acid (AA) sequence homology to the sequence of S. mutans led researchers to believe that it could use a CitMHS transporter to access iron. In fact, this was shown not to be the case. The system was shown to be a functional CitM (B. subtilis) homolog, with metals with larger ionic radii, such as Ca2+, Sr2+, Cd2+, and Pb2+, but not Fe2+ or Fe3+, involved in transport. This unpredictability clearly demonstrates our limited understanding of these systems, which drove us to investigate the postulated transporter in Streptomyces coelicolor. The genome of S. coelicolor was sequenced in 2002 (4). This effort identified an unprecedented number of genes encoding membrane-spanning transporters and gene sets that encode enzymes for utilizing complex nutrients. Among the possible functions of these genes was the putative ability to take up citrate in a complexed, metal-bound form. A comprehensive phylogenetic tree of the CitMHS family reported by Blancato et al. shows this S. coelicolor CitMHS member on a branch well separated from those systems investigated to date (5). The transporters on S. coelicolor's branch actually share only between 35 and 45% AA sequence homology with those transporters investigated to date, compared to the 60 to 83% AA homology between the B. subtilis, E. faecalis, and S. mutans transporters. Given the importance of S. coelicolor in the environment, the weak AA sequence homology (sequence identity does not warrant the same substrate specificity) (1) to all CitMHS transporters studied to date, and the fact that it may serve as a model organism for disease-causing members of the actinomycete family, such as M. tuberculosis, we set out to investigate this CitMHS family member and evaluate its contribution, if any, to the pathogenicity of bacteria by iron acquisition. To that end, we have functionally characterized metal-citrate transport in S. coelicolor and successfully performed heterologous expression in Escherichia coli of the transporter (designated here S. coelicolor Cit [CitSc]).