Abstract
Comparative genomic studies of the bacterial MFS-type copper importer CcoA, required for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis, revealed a widespread CcoA-like transporters (CalT) family, containing the conserved CcoA Cu-binding MxxxM and HxxxM motifs. Surprisingly, this family also included the RfnT-like proteins, earlier suggested to transport riboflavin. However, presence of the Cu-binding motifs in these proteins raised the possibility that they might be Cu transporters. To test this hypothesis, the genomic context of the corresponding genes was examined, and three of such genes from Ochrobactrum anthropi, Rhodopseudomonas palustris and Agrobacterium tumefaciens were expressed in Escherichia coli (ΔribB) and Rhodobacter capsulatus (ΔccoA) mutants. Copper and riboflavin uptake abilities of these strains were compared with those expressing R. capsulatus CcoA and Rhizobium leguminosarum RibN as bona fide copper and riboflavin importers, respectively. Overall data demonstrated that the “RfnT-like” CalT proteins are unable to efficiently transport riboflavin, but they import copper like CcoA. Nevertheless, even though expressed and membrane-localized in a R. capsulatus mutant lacking CcoA, these transporters were unable to accumulate Cu or complement for cbb3-Cox defect. This lack of functional exchangeability between the different subfamilies of CalT homologs suggests that MFS-type bacterial copper importers might be species-specific.
Highlights
The first bacterial copper (Cu) importer of MFS-type, CcoA (RCC02192) was discovered in the facultative photosynthetic model organism Rhodobacter capsulatus[12,13] and since has become the prototype of the newly www.nature.com/scientificreports/
We examined the genomic context of CcoA-like transporters (CalT) genes, and expressed three riboflavin transporters (RfnT)-like CalT homologs from O. anthropi, A. tumefaciens and Rhodopseudomonas palustris in E. coli ΔribB and R. capsulatus ΔccoA mutants, and investigated their ability to transport Cu or riboflavin
The vast majority of proteins from each taxonomically distinct subfamily of CalT contain the motifs MxxxM in TM7 and HxxxM in TM8, which are required for Cu uptake and cbb3-Cox biogenesis in R. capsulatus[16]
Summary
The first bacterial copper (Cu) importer of MFS-type, CcoA (RCC02192) was discovered in the facultative photosynthetic model organism Rhodobacter capsulatus[12,13] and since has become the prototype of the newly www.nature.com/scientificreports/. We performed a phylogenomic analysis to determine whether RfnT defines a functionally different subgroup of the CalT family, with distinct substrate specificity To this end, we examined the genomic context of CalT genes, and expressed three RfnT-like CalT homologs from O. anthropi (calT-O: OANT_22955, formerly called RfnT19), A. tumefaciens (calT-A: ATU_1173) and Rhodopseudomonas palustris (calT-R: TX73-RS24555) in E. coli ΔribB and R. capsulatus ΔccoA mutants, and investigated their ability to transport Cu or riboflavin. The findings showed that the conserved MxxxM and HxxxM motifs are strong predictors of Cu-transport activity, and suggested that members of the CalT family may exhibit species-specificity for Cu uptake, illustrating the ability of different organisms to use dedicated Cu uptake and delivery pathways to channel Cu to target proteins
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