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.

Energy-coupling factor (ECF) transporters for uptake of vitamins and transition-metal ions into prokaryotic cells share a common architecture consisting of a substrate-specific integral membrane protein (S), a transmembrane coupling protein (T) and two cytoplasmic ATP-binding-cassette-family ATPases. S components rotate within the membrane to expose their binding pockets alternately to the exterior and the cytoplasm. In contrast to vitamin transporters, metal-specific systems rely on additional proteins with essential but poorly understood functions. CbiN, a membrane protein composed of two transmembrane helices tethered by an extracytoplasmic loop of 37 amino-acid residues represents the auxiliary component that temporarily interacts with the CbiMQO2 Co2+ transporter. CbiN was previously shown to induce significant Co2+ transport activity in the absence of CbiQO2 in cells producing the S component CbiM plus CbiN or a Cbi(MN) fusion. Here we analyzed the mode of interaction between the two protein domains. Any deletion in the CbiN loop abolished transport activity. In silico predicted protein-protein contacts between segments of the CbiN loop and loops in CbiM were confirmed by cysteine-scanning mutagenesis and crosslinking. Likewise, an ordered structure of the CbiN loop was observed by electron paramagnetic resonance analysis after site-directed spin labeling. The N-terminal loop of CbiM containing three of four metal ligands was partially immobilized in wild-type Cbi(MN) but completely immobile in inactive variants with CbiN loop deletions. Decreased dynamics of the inactive form was also detected by solid-state nuclear magnetic resonance of isotope-labeled protein in proteoliposomes. In conclusion, CbiM-CbiN loop-loop interactions facilitate metal insertion into the binding pocket.

Energy-coupling factor (ECF) transporters mediate the uptake of micronutrients in prokaryotes. They consist of two ATP-binding-cassette family ATPases, a transmembrane coupling protein (T component) and a substrate-binding membrane protein (S component). ECF transporters for Co2+ and Ni2+ ions have one or two additional proteins with extracytoplasmic regions but poorly understood function. Homologs of T components with a predicted localization in plastids are widespread in plants but their physiological role is unclear. S components in eukaryotes are very rare and restricted to biotin-specific variants. Apart from a potential contribution to the export of flavins to serve the assembly of extracytoplasmic electron transfer chains, ECF transporters function as importers.

The mechanism of energy-coupling factor (ECF) transporters, a special type of ATP-binding-cassette importers for micronutrients in prokaryotes, is a matter of controversial discussion. Among subclass II ECF transporters, a single ECF interacts with several substrate-binding integral membrane proteins (S units) for individual solutes. Release and catch of the S unit, previously observed experimentally for a subclass II system, was proposed as the mechanism of all ECF transporters. The BioM2NY biotin transporter is a prototype of subclass I systems, among which the S unit is dedicated to a specific ECF. Here we simulated the transport cycle using purified BioM2NY in detergent solution. BioM2NY complexes were stable during all steps. ATP binding was a prerequisite for biotin capture and ATP hydrolysis for subsequent biotin release. The data demonstrate that S units of subclass I ECF transporters do not have to dissociate from holotransporter complexes for high-affinity substrate binding, indicating mechanistic differences between the two subclasses.

Background: Substrate-binding integral membrane proteins of ECF transporters are predicted to undergo reversible rotation during the transport cycle. Results: Capture and release of biotin by a nanodisc-embedded ECF transporter depended on ATP-induced subunit reorientations. Conclusion: ECF transporters mediate vitamin translocation by turning their substrate-specific components within the membrane. Significance: Individual steps of the transport cycle are highlighted by biochemical and biophysical techniques. Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [3H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Biotin is an essential cofactor of carboxylase enzymes in all kingdoms of life. The vitamin is produced by many prokaryotes, certain fungi, and plants. Animals depend on biotin uptake from their diet and in humans lack of the vitamin is associated with serious disorders. Many aspects of biotin metabolism, uptake, and intracellular transport remain to be elucidated. In order to characterize the activity of novel biotin transporters by a sensitive assay, an Escherichia coli strain lacking both biotin synthesis and its endogenous high-affinity biotin importer was constructed. This strain requires artificially high biotin concentrations for growth. When only trace levels of biotin are available, it is viable only if equipped with a heterologous high-affinity biotin transporter. This feature was used to ascribe transport activity to members of the BioY protein family in previous work. Here we show that this strain together with its parent is also useful as a diagnostic tool for wide-concentration-range bioassays.

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.

Biotin is an essential cofactor of carboxylase enzymes in all kingdoms of life. The vitamin is produced by many prokaryotes, certain fungi, and plants. Animals depend on biotin uptake from their diet and in humans lack of the vitamin is associated with serious disorders. Many aspects of biotin metabolism, uptake, and intracellular transport remain to be elucidated. In order to characterize the activity of novel biotin transporters by a sensitive assay, an Escherichia coli strain lacking both biotin synthesis and its endogenous high-affinity biotin importer was constructed. This strain requires artificially high biotin concentrations for growth. When only trace levels of biotin are available, it is viable only if equipped with a heterologous high-affinity biotin transporter. This feature was used to ascribe transport activity to members of the BioY protein family in previous work. Here we show that this strain together with its parent is also useful as a diagnostic tool for wide-concentration-range bioassays.

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 Thermoanaerobacter 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-terminus. 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 Cu(2+)- and Ni(2+)-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 Co(2+). Indeed, the structure of TtNikM2 containing a bound Co(2+) 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 form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [(3)H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane.

BioMNY (Rhodobacter capsulatus) is an ATP dependent, ECF-type biotin importer, consisting of two different transmembrane domains BioY, BioN and ATPase cassettes BioM. The organization and stoichiometry of the transporter subunits is still a matter of debate (Erkens, 2011; Berntsson, 2012; Erkens, 2012). To obtain insights into the stoichiometry of the transporter components we used spectrometric, lifetime-based HeteroFRET (Finkenwirth, 2010) and static anisotropy-based HomoFRET (Kirsch, 2012) approaches. To this aim, certain transporter components, tagged with a fluorescence donor or acceptor, were co-expressed in recombinant E. coli. The measurements led to the postulation of a BioM2N?Y2 stoichiometry in living bacteria (in accordance with Neubauer, 2009, 2011). Recently, we expanded the Hetero and HomoFRET approach form the spectroscopic to the microscopic level (Ziomkowska, 2012). The use of imaging techniques for microbial cells has the same advantages as for mammalian cells: expression level and localization of the protein can be validated in a single cell during the measurement. Our experiments show that the spectroscopic data can be reproduced in imaging experiments and will be used for the ongoing research. Nevertheless, the orientation of the probe has to be taken into account for anisotropy microscopy. To the best of our knowledge, we present the first FRET-Imaging approach on living bacteria, a tool likely to be used for interaction studies in any E. coli expression system.

Energy-coupling factor transporters are a large group of importers for trace nutrients in prokaryotes. The in vivo oligomeric state of their substrate-specific transmembrane proteins (S units) is a matter of debate. Here we focus on the S unit BioY of Rhodobacter capsulatus, which functions as a low-affinity biotin transporter in its solitary state. To analyze whether oligomerization is a requirement for function, a tail-to-head-linked BioY dimer was constructed. Monomeric and dimeric BioY conferred comparable biotin uptake activities on recombinant Escherichia coli. Fluorophore-tagged variants of the dimer were shown by fluorescence anisotropy analysis to oligomerize in vivo. Quantitative mass spectrometry identified biotin in the purified proteins at a stoichiometry of 1:2 for the BioY monomer and 1:4 (referring to single BioY domains) for the dimer. Replacement of the conserved Asp164 (by Asn) and Lys167 (by Arg or Gln) in the monomer and in both halves of the dimer inactivated the proteins. The presence of those mutations in one half of the dimers only slightly affected biotin binding but reduced transport activity to 25% (Asp164Asn and Lys167Arg) or 75% (Lys167Gln). Our data (i) suggest that intermolecular interactions of domains from different dimers provide functionality, (ii) confirm an oligomeric architecture of BioY in living cells, and (iii) demonstrate an essential role of the last transmembrane helix in biotin recognition.

Förster resonance energy transfer (FRET) identifies the co-localization of fluorescent macromolecules on a nanometer scale. FRET between nonidentical and identical fluorophores is called HeteroFRET or HomoFRET, respectively. We applied FRET techniques to analyze the oligomeric state of the core component (BioY) of a bacterial biotin transporter. BioY was fused to fluorescent proteins suitable for Hetero- and HomoFRET analyses. Spectrometric and imaging experiments with living cells strongly support a model with oligomers of BioY as the functional state of the core unit.

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.

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.

BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor-acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor-acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.