Comparative and functional genomic analysis of prokaryotic nickel and cobalt uptake transporters: evidence for a novel group of ATP-binding cassette transporters

Abstract

The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B(12) riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B(12) biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.

FIG. 1.

Conservation of the NikR regulon in bacteria and archaea. (A) Sequence logos for the predicted NikR-binding signals in various taxonomic groups. (B) Maximum likelihood phylogenetic tree of the NikR proteins. All NikR-binding sites identified in the genomes that encode NikR from a certain group (marked by background gray) were used for construction of the sequence logo for the corresponding group. Candidate NikR-binding sites were not identified in only a few archaeal genomes encoding NikR (unmarked proteins on the phylogenetic tree). Three surface residues of the N-terminal beta sheets involved in DNA recognition are shown in boxes.

FIG. 2.

Genomic organization of bacterial genes encoding nickel- or coenzyme B₁₂-regulated outer membrane receptors. Black circles and triangles indicate candidate NikR-binding sites and B₁₂ riboswitch regulatory elements, respectively.

FIG. 3.

Topology prediction for nickel and cobalt ABC transporters of the CbiMNQO/NikMNQO family. Homologous protein components of different transport systems are colored in the same way. Membrane-bound components are shown by rectangles with predicted transmembrane helices shown by cylinders. Putative ATPase components are shown by pink ovals. Periplasmic domains of NikL and NikK are shown by turquoise and yellow ovals, respectively. The fifth transmembrane helix is not conserved in some CbiQ/NikQ proteins and is shown in dotted lines. NikM and NikN components are fused to form a single protein in many cases. The illustrated physical interaction between O and Q components is speculative. The red arrow indicates a putative cleavage site (AnAMH) by bacterial signal peptidase I in all CbiM proteins that contain transmembrane helix 0.

FIG. 4.

Maximum likelihood phylogenetic tree of the M components of Cbi/Nik transport systems. Genes predicted to be regulated by the nickel repressor NikR and coenzyme B₁₂ riboswitch are in blue and yellow background colors, respectively. Genomic colocalizations of cbi/nik genes with genes encoding Ni-dependent enzymes and B₁₂ biosynthesis proteins are indicated by blue and yellow dots, respectively. The color of each of the lines indicates the subunit composition of the respective nickel/cobalt transport system. Dashed lines indicate transporters containing fused M and N subunits.

FIG. 5.

⁵⁷Co²⁺ (empty circles) and ⁶³Ni²⁺ (filled circles) uptake of recombinant E. coli cells expressing cbiMNQO from Salmonella enterica serovar Typhimurium (St) or Rhodobacter capsulatus (Rc) or nik(MN)QO from R. capsulatus. Control cells contained an empty pBluescript II KS+ vector. Cells were grown in the presence of either of the radiolabeled metal salts. The cellular metal content was determined by liquid scintillation counting. The inset in the upper right panel illustrates RcCbiMNQO-mediated nickel-uptake activity at a refined scale.

FIG. 6.

Cobalt uptake of recombinant E. coli expressing S. enterica serovar Typhimurium cbiMNQO (white bars), cbiMNQ (diagonally hatched bars), cbiMN (horizontally lined bars), or cbiM (gray bars) or containing an empty vector (black bars).