RESUMEN
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.
Asunto(s)
Canavalia/enzimología , Inhibidores Enzimáticos/farmacología , Klebsiella pneumoniae/metabolismo , Níquel/metabolismo , Urea/metabolismo , Ureasa/antagonistas & inhibidores , Transporte Biológico , Inhibidores Enzimáticos/clasificación , Hidrólisis , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobalto/metabolismo , Proteínas de Escherichia coli/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Sitios de Unión , Proteínas de Transporte de Catión/química , Proteínas de Escherichia coli/química , Unión ProteicaRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Bacterias/metabolismo , Cobalto/metabolismo , Níquel/metabolismo , Vitaminas/metabolismo , Transporte Biológico/fisiología , Biotina/metabolismo , Membrana Celular/metabolismo , Modelos Moleculares , Plantas/metabolismo , Conformación ProteicaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Rhodobacter capsulatus/química , Simportadores/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo/fisiología , Estabilidad Proteica , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
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 [(3)H]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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biotina/metabolismo , Rhodobacter capsulatus/metabolismo , Simportadores/química , Simportadores/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Conformación Proteica , Rhodobacter capsulatus/química , Rhodobacter capsulatus/genética , Simportadores/genéticaRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Proteínas Bacterianas/fisiología , Cobalto/metabolismo , Níquel/metabolismo , Transporte Biológico Activo , Membrana Celular/metabolismo , Enterobacter aerogenes/enzimología , Escherichia coli/enzimología , Rhodobacter capsulatus/genética , Ureasa/metabolismoRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Cobalto/metabolismo , Níquel/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Secuencias de Aminoácidos , Sitios de Unión , Cobalto/química , Cristalografía por Rayos X , Iones/química , Simulación de Dinámica Molecular , Níquel/química , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Teoría Cuántica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato , Thermoanaerobacter/metabolismo , Vitaminas/química , Vitaminas/metabolismoRESUMEN
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.
Asunto(s)
Bioensayo/métodos , Biotina/genética , Biotina/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Simportadores/genética , Simportadores/farmacología , Biotina/análisis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Simportadores/análisisRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Transporte Biológico Activo , Biotina/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Recombinación Genética , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/genética , Escherichia coli K12/genética , Escherichia coli K12/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , MutaciónRESUMEN
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.
Asunto(s)
Rhodobacter capsulatus/metabolismo , Simportadores/química , Simportadores/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Transporte Biológico , Biotina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Simportadores/metabolismo , Cisteína/química , Ensayo de Cambio de Movilidad Electroforética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Simportadores/químicaRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Variación Genética , Archaea/fisiología , Bacterias/patogenicidad , Modelos Biológicos , Modelos MolecularesRESUMEN
ECF-class transporters comprise abundant importers for micronutrients such as vitamins and transition-metal ions, and for intermediates of salvage pathways in bacteria and archaea. They are composed of ABC ATPases (A units), a conserved transmembrane protein (T unit) and a substrate-specific transmembrane protein (S unit or core transporter). Here we analyzed the function of an ECF-type Co(2+) transporter (CbiMNQO) and, in particular, the derived bipartite S unit CbiMN. CbiMN was characterized as the minimal unit that functions as a Co(2+) transporter. Neither the solitary CbiM nor a tripartite CbiMQO complex was active, indicating an essential role for CbiN. CbiN was loosely bound in CbiMNQO and CbiMN complexes, and did not copurify with its partners. Generating a contiguous reading frame resulted in a Cbi(MN) fusion protein that displayed Co(2+)-transport activity and interacted with CbiQO in vivo. Sixteen variants of Cbi(MN) with modifications in the strongly conserved N-terminal stretch of ten amino-acid residues were constructed and analyzed for transport activity. The results indicate that the length and sequence of this region are critical for functioning of the core transporter. Specifically, they point to essential roles of His2 and the distance of His2 to the amino group of the peptide chain in metal recognition.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Cobalto/metabolismo , Rhodobacter capsulatus/enzimología , Escherichia coli/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rhodobacter capsulatus/genéticaRESUMEN
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.
Asunto(s)
Escherichia coli/química , Simportadores/análisis , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Multimerización de Proteína , Subunidades de Proteína/análisis , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Simportadores/metabolismoRESUMEN
Energy-coupling factor (ECF) transporters, a recently discovered class of importers of micronutrients, are composed of a substrate-specific transmembrane component (S component) and a conserved energy-coupling module consisting of a transmembrane protein (T component) and pairs of ABC ATPases (A proteins). Based on utilization of a dedicated (subclass I) or shared (subclass II) energy-coupling module, ECF systems fall into two subclasses. The T components are the least-characterized proteins of ECF importers, and their function is essentially unknown. Using RcBioN and LmEcfT, the T units of the subclass I biotin transporter (RcBioMNY) of a gram-negative bacterium and of the subclass II folate, pantothenate, and riboflavin transporters of a lactic acid bacterium, respectively, we analyzed the role of two strongly conserved short motifs, each containing an arginine residue. Individual replacement of the two Arg residues in RcBioN reduced ATPase activity, an indicator of the transporter function, by two-thirds without affecting the modular assembly of the RcBioMNY complex. A double Arg-to-Glu replacement destroyed the complex and abolished ATPase activity. The corresponding single mutation in motif II of LmEcfT, as well as a double mutation, led to loss of the T unit from the subclass II ECF transporters and inactivated these systems. A single Arg-to-Glu replacement in motif I, however, abolished vitamin uptake activity without affecting assembly of the modules. Our results indicate that the conserved motif I in T components is essential for intramolecular signaling and, in cooperation with motif II, for subunit assembly of modular ECF transporters.
Asunto(s)
Arginina/química , Proteínas Portadoras/química , Metabolismo Energético/fisiología , Escherichia coli/metabolismo , Vitaminas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiologíaRESUMEN
The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Vitaminas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Clonación Molecular , Cobalto/metabolismo , Biología Computacional , Bases de Datos de Proteínas , Genoma , Leuconostoc/genética , Proteínas de Transporte de Membrana/clasificación , Proteínas de Transporte de Membrana/genética , Níquel/metabolismo , Mapeo RestrictivoRESUMEN
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 alpha-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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biotina/metabolismo , Células Procariotas/metabolismo , Rhodobacter capsulatus/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Biología Computacional , Escherichia coli , Genómica , Cinética , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Genoma Bacteriano , Aerobiosis , Anaerobiosis , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Transporte Biológico , Carbono/metabolismo , Cromosomas Bacterianos , Hidroxibutiratos/metabolismo , Datos de Secuencia Molecular , Poliésteres/metabolismoRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Cobalto/metabolismo , Genómica , Níquel/metabolismo , Proteobacteria/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cobamidas , Biología Computacional , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Filogenia , Proteobacteria/genética , Proteínas Represoras/metabolismoRESUMEN
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.