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Since their discovery in the 1960s as ‘osmotic shock-sensitive' transporters, a plethora of so-called binding protein-dependent (canonical) ATP-binding cassette (ABC) importers has been identified in bacteria and archaea. Their cellular functions go far beyond the uptake of nutrients. Canonical ABC importers play important roles in the maintenance of cell integrity, responses to environmental stresses, cell-to-cell communication and cell differentiation and in pathogenicity. A new class of abundant micronutrient importers, the ‘energy-coupling factor' (ECF) transporters, was originally identified by functional genomics. ABC ATPases are an integral part of both canonical ABC and ECF importers. Fundamental differences include the modular architecture and the independence of ECF systems of extracytoplasmic solute-binding proteins. This review describes the roles of both types of transporters in diverse physiological processes including pathogenesis, points to the differences in modular assembly and depicts their common traits.

The H 2 -oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H 2 and CO 2 as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[ R -(–)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.

BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one-third of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the α-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters.

The energy-coupling factor （ECF） transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component （S component） of an ECF-type nickel transporter from Thermo- anaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments （TSs）, TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-termi- nus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu2＋- and Ni2＋-binding （ATCUN） motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co2＋. Indeed, the structure of TtNikM2 containing a bound Co2＋ ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters.

Energy-coupling factor (ECF) transporters are a huge group of micronutrient importers in prokaryotes. They are composed of a substrate-specific transmembrane protein (S component) and a module consisting of a moderately conserved transmembrane protein (T component) and two ABC ATPase domains (A components). Modules of A and T units may be dedicated to a specific S component or shared by many different S units in an organism. The mode of subunit interactions in ECF transporters is largely unknown. BioMNY, the focus of the present study, is a biotin transporter with a dedicated AT module. It consists of the S unit BioY, the A unit BioM and the T unit BioN. Like all T units, BioN contains two three-amino-acid signatures with a central Arg residue in a cytoplasmic helical region. Our previous work had demonstrated a central role of the two motifs in T units for stability and function of BioMNY and other ECF transporters. Here we show by site-specific crosslinking of pairs of mono-cysteine variants that the Ala-Arg-Ser and Ala-Arg-Gly signatures in BioN are coupling sites to the BioM ATPases. Analysis of 64 BioN-BioM pairs uncovered interactions of both signatures predominantly with a segment of ~13 amino acid residues C-terminal of the Q loop of BioM. Our results further demonstrate that portions of all BioN variants with single Cys residues in the two signatures are crosslinked to homodimers. This finding may point to a dimeric architecture of the T unit in BioMNY complexes.

The nickel-dependent urease enzyme is responsible for the hydrolysis of urea to ammonia and carbon dioxide. A number of bacteria produce urease (ureolytic bacteria) and are associated with various infectious diseases and ammonia emissions from agriculture. We report the first comprehensive comparison of the inhibition of urease activity by compounds analysed under the same conditions. Thus, 71 commercially available compounds were screened for their anti-ureolytic properties against both the ureolytic bacterium Klebsiella pneumoniae and purified jack bean urease. Of the tested compounds, 30 showed more than 25% inhibition of the ureolytic activity of Klebsiella pneumoniae or jack bean urease, and among these, carbon disulfide, N-phenylmaleimide, diethylenetriaminepentaacetic acid, sodium pyrrolidinedithiocarbamate, 1,2,4-butanetricarboxylic acid, tannic acid, and gallic acid have not previously been reported to possess anti-ureolytic properties. The diverse effects of metal ion chelators on ureolysis were investigated using a cellular nickel uptake assay. Ethylenediaminetetraacetic acid (EDTA) and dimethylglyoxime (DMG) clearly reduced the nickel import and ureolytic activity of cells, oxalic acid stimulated nickel import but reduced the ureolytic activity of cells, 1,2,4-butanetricarboxylic acid strongly stimulated nickel import and slightly increased the ureolytic activity of cells, while L-cysteine had no effect on nickel import but efficiently reduced the ureolytic activity of cells.

Nickel/cobalt permeases (NiCoTs, TC 2.A.52) are a rapidly growing family of structurally related membrane transporters whose members are found in Gram-negative and Gram-positive bacteria, in thermoacidophilic archaea, and in fungi. Previous studies have predicted two subclasses represented by HoxN of Ralstonia eutropha, a selective nickel transporter, and by NhlF of Rhodococcus rhodochrous, a nickel and cobalt transporter that displays a preference for the Co ion. In the present study, NiCoT genes of five Gram-negative bacteria and one Gram-positive bacterium were cloned and heterologously expressed in Escherichia coli. Based on substrate preference in metal-accumulation assays with the recombinant strains, two of the novel NiCoTs were assigned to the NhlF class. The remaining four NiCoTs belong to a yet unrecognized, third class. They transport both the nickel and the cobalt ion but have a significantly higher capacity for nickel. The observed substrate preferences correlate in many cases with the genomic localization of NiCoT genes adjacent to regions encoding nickel- or cobalt-dependent enzymes or enzymes involved in cobalamin biosynthesis. Alignment of 23 full-length NiCoT sequences and comparison with the available experimental data predict that substrate specificity of NiCoTs is an adaptation to specific transition metal requirements in various organisms from different taxa.

Energy-coupling factor (ECF) transporters form a distinct group of ABC-type micronutrient importers in prokaryotes that do not contain extracytoplasmic, soluble substrate-binding proteins. Instead, they consist of a transmembrane substrate-specific S component that interacts with a module composed of a moderately conserved transmembrane (T) component and ABC ATPases. The majority of S components is considered to act as high-affinity binding proteins that strictly depend on their cognate T and ATPase units for transport activity. For a fraction of biotin-specific S units, however, transport activity was demonstrated in their solitary state. Here, we compared the activities of nickel- and cobalt-specific ECF transporters in the presence and absence of their T and ATPase units. Accumulation assays with radioactive metal ions showed that the truncated transporters led to approx. 25 % of cell-bound radioactivity compared to the holotransporters. Activity of urease, an intracellular nickel-dependent enzyme, was used as a reporter and clearly indicated that the cell-bound radioactivity correlates with the cytoplasmic metal concentration. The results demonstrate that S units of metal transporters not only bind their substrates on the cell surface but mediate transport across the membrane, a finding of general importance on the way to understand the mechanism of ECF transporters.

Nickel/cobalt transporters (NiCoTs), a family of secondary metal transporters in prokaryotes and fungi, are characterized by an eight-transmembrane-domain (TMD) architecture and mediate high-affinity uptake of cobalt and/or nickel ions into the cells. One of the strongly conserved regions within the NiCoTs is the signature sequence RHA(V/F)DADHI within TMD II. This stretch of amino acid residues plays an important role in the affinity, velocity and specificity of metal transport. Some relatives of the NiCoTs, named HupE, UreJ and UreH, contain a similar signature sequence and are encoded within or adjacent to [NiFe] hydrogenase or urease operons, or elsewhere in the genome of many prokaryotes. HupE and UreH from Rhodopseudomonas palustris CGA009 and UreJ from Cupriavidus necator H16 were shown to mediate Ni(2+) transport upon heterologous production in E. coli. Other variants of NiCoTs are found in many marine cyanobacteria and in plants. The cyanobacterial proteins are encoded by a segment adjacent to the genes for [Ni] superoxide dismutase and a corresponding putative maturation peptidase. The plant proteins contain N-terminal sequences resembling bipartite transit peptides of thylakoid lumenal and thylakoid integral membrane precursor proteins; expression of a YFP-fusion protein in transfected leaf cells is consistent with targeting of this protein to the plastid, but the function of the plant gene product has yet to be demonstrated.