This chapter summarizes the current knowledge on canonical ABC import systems involved in the uptake of macronutrients, trace elements, vitamins and polyamines.
Carbon and energy sources
SBP-dependent ABC transporters mediating the uptake of a diverse range of carbohydrates for carbon and energy metabolism cluster primarily in the CUT1 and CUT2 families of the TC classification system (Saier, 2000; Schneider, 2001). The ‘earmark’ of CUT2 family members is the primary structure of their NBDs, which appear to be a ‘natural’ fusion of two ABC domains (Schneider, 2001). Some carbohydrate transporters of hyperthermophilic Archaea, for example Pyrococcus furiosus (Koning et al., 2001) and Sulfolobus solfataricus (Elferink et al., 2001), Thermotoga maritima (a hyperthermophilic bacterium) (Conners et al., 2005; Nanavati et al., 2006) and of the root nodule-forming bacterium Rhizobium meliloti are also found within the PepT subfamily, otherwise comprising transporters for peptides, opines and nickel ions.
While the per se existence of mono- and disaccharides in nature is rather limited (e.g. fruits, milk, sugar-rich diet of animals, including humans), the major sources of sugars that can be utilized by prokaryotes are plant- and fungi-derived polysaccharides, including starch, cellulose, xylan, pectin and chitin. Thus, most prokaryotes that thrive in soil, aquatic environments, sewage digesters or the digestive tracts of animals secrete hydrolases for the degradation of biopolymers. The resulting mono-, di- and oligosaccharides are then internalized primarily due to the action of specific ABC transporters.
Maltose, maltodextrins, cyclodextrins. The maltose/maltodextrin transporter MalEFGK2 of E. coli/S. Typhimurium, by far the best-characterized member of the CUT1 family that also serves as a model for ABC transporters in general (see The ‘alternating access’-model of transport), enables the facultative anaerobes at the entry of the large intestine to feed on sugars formed by the cleavage of starch from the animals' diet in the stomach and that escaped adsorption in the small intestine (Flint et al., 2008). Quantitatively predominant bacteria in the large intestine such as Bifidobacterium have multiple gene clusters encoding putative oligosaccharide ABC transporters in their genomes (Schell et al., 2002).
The receptor, MalE, binds maltose and maltooligosaccharides up to seven glucose units (Boos & Shuman, 1998), which are delivered to the MalFGK2 complex for translocation to the cytoplasm. In contrast, reduced or oxidized maltodextrins or β-cyclodextrin bind to MalE, but the resulting liganded receptor fails to initiate the transport cycle, due to a different binding mode (Hall et al., 1997a, b). The NBD MalK and MalFGK2 were purified and extensively characterized in liposomes (reviewed in Schneider, 2003; Davidson & Chen, 2004). Crystal structures were resolved of MalE complexed with various substrates (Quiocho et al., 1997), of the MalK dimer (reviewed in Davidson & Chen, 2004) and the complete transporter in the apo form in the absence of MalE (Khare et al., 2009) and in an ATP-bound form with tightly associated MalE (Oldham et al., 2007). The C-terminal extension of the MalK, the NBD of the maltose/maltodextrin transporter from E. coli/Salmonella, binds MalT, the positive transcriptional regulator of the mal regulon, thereby preventing its activation through binding of ATP and maltotriose and subsequent oligomerization (Joly et al., 2004; Richet et al., 2005). It is proposed that substrate availability is sensed through the transporter, which, in the idling mode, binds MalT and thereby represses mal gene transcription. In the presence of a substrate, however, transport activity is switched on, i.e. ATP is hydrolyzed at the MalK subunits, thus causing the release of MalT and subsequent induction of maltose-regulated gene expression (Boos & Böhm, 2000). The maltose transporter is also involved in a second regulatory process called ‘inducer exclusion’, which is part of the global carbon regulation in enteric bacteria. Here, in the presence of the preferred carbon source, glucose, the transport of inducer molecules for alternative metabolic pathways is prevented. This is achieved by inhibition of the respective transport systems via a component of the glucose transporter, the dephosphorylated enzyme IIAGlc of the phosphoenolpyruvate phosphotransferase system (PTS) (Postma et al., 1996). In the case of the maltose transporter, enzyme IIAGlc binds predominantly to the C-terminal extension of the MalK subunits, thereby inhibiting ATP hydrolysis (reviewed in Schneider, 2003). Although the precise mechanism of inhibition is still unknown, the structure of the MalK dimer suggests binding sites for enzyme IIAGlc on the C-terminal domain of one monomer and the N-terminal domain of the opposing monomer (Samanta et al., 2003). Although an additional C-terminal domain of their NBDs is shared by all members of the CUT1 family, in the majority of cases, nothing is known on the possible regulatory processes involved.
Other related transporters discriminate between maltose and maltooligosaccharides by specific properties of their cognate receptors. Thus, the maltotriose porter of Thermus thermophilus does not recognize the disaccharide maltose, while the MalEFGK2 transport system of Thermococcus litoralis accepts maltose and trehalose, but no longer dextrins. On the other hand, trehalose is not a substrate of the E. coli maltose/maltodextrin porter (Cuneo et al., 2009b). Comparison of crystal structures of these proteins in complex with the cognate substrates revealed the presence of four subsites that bind individual glucose rings (Samanta et al., 2003; Cuneo et al., 2009b).
An ABC transporter homologous to maltose/maltodextrin porters of the CUT1 family also plays a key role in the proposed ‘carbophor’ function of the pseudo-maltotetraose acarbose produced by bacteria from the genera Actinoplanes and Streptomyces (reviewed in (Wehmeier & Piepersberg, 2004). Acarbose, which is also a substrate of the E. coli maltose transporter (Brunkhorst et al., 1999), consists of an unsaturated C7 cyclitol bound via an imino bridge to 4-amino-4,6-dideoxyglucose (together named acarviosine) to which a maltose moiety is attached. According to this hypothesis, acarbose is synthesized and secreted into the environment, where, due to the action of a transferase, the acarviosyl group is hooked to maltotriose or other oligosaccharides. These are taken up by the GacHFG-MsiK ABC transporter, thereby resulting in a net gain in carbon and energy. Crystal structures and biochemical characterization of the binding protein GacH have demonstrated that longer acarbose homologs are in fact ligands of the protein, thereby corroborating this notion (Vahedi-Faridi et al., 2010).
Two CUT1 family members with strong sequence similarities to the E. coli maltose transport proteins, but distinctive substrate specificities, have been described in Streptococcus mutans, which is central to dental caries in humans. While the MsmEFGK system transports raffinose, melibiose and stachyose, the MalXFGK transporter is specific for maltodextrins. Maltose is a poor substrate for both systems. Mutations affecting the binding proteins, MsmE and MalX, respectively, were shown to cause the respective defects in sugar utilization (Webb et al., 2008). These results somewhat contradict a study by Kilic et al. (2007), who reported overlapping substrate specificities for both systems. However, mutating either one of the ATPase subunits, MsmK and MalK, had no phenotype, suggesting that the remaining NBD is shared by both systems (Webb et al., 2008).
Similar conclusions were drawn previously in the case of the MsiK protein, which assists in the uptake of several oligosaccharides through distinct transporters in Streptomyces (Hurtubise et al., 1995).
Bacteria such as Klebsiella oxytoca secrete cyclodextrin glycosyltransferases, which can cyclize linear maltodextrins first to α-cyclodextrins that are converted mainly to β-cyclodextrins. Utilization of cyclodextrins as carbon and energy source involves a cyclodextrin-ABC transport system of the CUT1 family (Fiedler et al., 1996). Crystal structures of a cyclodextrin-binding protein from Thermoactinomyces vulgaris (TvuCMBP) have been reported in complex with α-, β- and γ-cyclodextrins as well as with maltotetraose (Matsumoto et al., 2009). The structures revealed that TvuCMBP, in contrast to E. coli MalE, adopts the closed conformation with α-and β-cyclodextrins, but the open conformation with maltotetraose.
Cellooligodextrins. Extracellular degradation of cellulose results mainly in the production of cellotriose and cellobiose, which are internalized by ABC transport systems. In species of the genus Streptomyces that thrive in soil, an operon (cebEFG) was identified, encoding a solute-binding lipoprotein, CebE, and two membrane proteins: CebF and CebG (Schlösser et al., 1999). Transcription of the operon is induced by cellobiose. The gene for an NBD, as often observed for sugar ABC transporters in gram-positive bacteria, is not linked, but the MsiK protein was demonstrated to assist in cellobiose/cellotriose and maltose transport (Schlösser et al., 1997).
While the Ceb system of Streptomyces is a member of the CUT1 family, the hyperthermophilic archaea P. furiosus and S. solfataricus contain high-affinity cellobiose-uptake systems, homologous to oligopeptide transporters. This holds for quite a few sugar transporters of archaea and of the bacterium T. maritima, whereas the majority of systems belong to the CUT family (Albers et al., 2004). The P. furiosus Cbt transporter exhibits a Km for uptake of 175 nM and a Kd for solute binding of 45 nM. The binding protein, CbtA, which is anchored to the cytoplasmic membrane via an N-terminal transmembrane helix, has a broad substrate specificity, accepting cellodextrins up to five glucose units and laminaribiose, a degradation product of laminarin (1,3-β-d-glucan) (Koning et al., 2001). The crystal structure of a cellobiose-binding protein homologous to oligopeptide-binding proteins from T. maritima revealed a semi-specific recognition of the substrates. While the disaccharide cellobiose binds specifically at its nonreducing end, additional rings up to five (cellopentaose) are located in a solvent-filled groove. Interactions of the reducing end with the protein define the acceptable length of the substrate (Cuneo et al., 2009a).
The anaerobic thermophilic bacterium Clostridium thermocellum grows very efficiently on cellulose due to a multienzyme complex, the cellulosome. Five sugar ABC transporters were identified in the organism, which, by analysis of the binding properties of their cognate solute-binding lipoproteins, were demonstrated to display specificities for cellodextrins (CbpB-D), cellotriose (CbpA) and laminaribiose (Lbp), respectively (Nataf et al., 2009).
Hemicelluloses. Because of the tight association of hemicelluloses with cellulose fibrils in the plant cell wall, cellulose degraders also require hemicellulolytic activity. Hemicelluloses are a mixture of branched and linear polysaccharides. Endo-1,4-β-xylanases release short, modified oligoxylose units of two or more sugars from the polymer's backbone.
In Streptomyces thermoviolaceus, an ABC transporter, BxlEFG (a gene for an ATPase subunit is not linked), operates that takes up xylodextrins. The recombinant binding protein, BxlE, shows the highest affinity for xylobiose (Kd∼10−8 M) and xylotriose (Kd∼10−7 M). Repression of transcription by the regulator protein BxlR is relieved in the presence of xylobiose (Tsujibo et al., 2004). A similar system, XynEFG, was found in Geobacillus stearothermophilus. The purified binding protein, XynE, binds the substrates up to six xylose units with Kd values in the low μM range. Transcription of the operon is repressed by a specific regulator and activated in the presence of xylose by the regulator component of a two-component system (Shulami et al., 2007).
A putative ABC transporter proposed to mediate the uptake of methyl-α-d-glucuronosyl-xylotriose, a degradation product of side groups of xylan, was identified in Bacillus stearothermophilus T-6, but not characterized further (Shulami et al., 1999).
Pectin. The plant cell wall component pectin, the major matrix polysaccharide, consists of α-1,4-linked galacturonate residues forming the backbone of the molecule. Attached to it are neutral sugars such as rhamnose, arabinose, galactose or xylose. Pectin can be depolymerized by pectinases, leading to maceration.
In the plant pathogen Erwinia chrysanthemi, which causes soft rot disease on various plants, an ABC transporter, TogMNAB, besides others, internalizes oligogalacturonides and is involved in the chemotactic response toward these compounds (Hugouvieux-Cotte-Pattat et al., 2001). The structure of the binding protein component, TogB, of the homologous transporter from Yersinia enterocolitica was resolved and revealed selectivity for digalacturonic acid, especially for the 4,5-unsaturated form of the sugar (Abbott & Boraston, 2007).
Ramified regions of pectin contain galactan (β-1,4- or β-1,3-linked d-galactopyranose residues), which can also be utilized by E. chrysanthemi. The ganEFGK operon, encoding a transporter displaying a high similarity to the maltose transporter of E. coli, is required for growth on galactan as shown by mutational analysis (Delangle et al., 2007).
Rhamnose, a methyl-pentose sugar that is also found in the mucilage of legume plants, is taken up by the root–nodule symbiont Rhizobium leguminosarum bv. trifolii via the activity of the RhaSTPQ system, representing a binding protein, an ATPase and two membrane proteins, respectively (Richardson et al., 2004). Transport experiments revealed strict specificity for rhamnose as other pentoses, such as l-arabinose or l-fucose, failed to inhibit the uptake of radiolabeled rhamnose. The capability to internalize rhamnose is required for the bacteria to successfully compete for nodule occupancy. As an interesting yet to be unraveled feature, the transport activity is dependent on an active rhamnose kinase (Richardson & Oresnik, 2007). The rhamnose ABC transporter, like those for other pentoses found in numerous bacteria, for example arabinose or xylose, is a member of the CUT2 family (Schneider, 2001).
Chitin. Chitin, the major cell wall component of fungi, is a polymer of N-acetyl-d-glucosamine. Soil bacteria of the genus Streptomyces secrete chitinases and internalize the chitooligomers produced, chitobiose (N, N′-diacetylchitobiose) and N-acetylglucosamine (NAG), which can be utilized as carbon and energy sources.
The Ngc transporter, comprising the solute-binding lipoprotein, NgcE, and two membrane-integral subunits, NgcF and NgcG, of Streptomyces olivaceoviridis, is the only known uptake system for NAG besides a phosphotransferase system. NgcE specifically binds NAG and chitobiose (Saito & Schrempf, 2004). Analogous ABC transporters were found by bioinformatics in Silicibacter and Rhizobiales (Yang et al., 2006). In addition, Streptomyces contain a gene cluster, dasABC, that encodes a transporter for the uptake of chitobiose, which also plays a role in cell differentiation (Saito et al., 2007). The binding protein, DasA, exhibits the highest affinity for chitobiose (32 nM), but also accepts chitooligomers up to five NAG units with a reasonable affinity. Low-affinity binding was found for NAG (25 μM). The transporter is completed by the MsiK-ATPase as demonstrated by mutational analysis (Saito et al., 2008).
Chitin is also produced in massive amounts in marine environments by crustaceans. Gene clusters for chitin utilization including genes that encode an ABC importer were found in Vibrio chlolerae and Vibrio furnissii (Li & Roseman, 2004).
Alginate. Sphingomonas sp. A1 can grow on alginate, a linear polymer composed of α-l-guluronate β-d-mannuronate (polymerization grade >100), and produced by brown seaweed and certain bacteria. Strikingly, and unlike other biopolymers, alginate is not degraded by secreted hydrolases before internalization. This is achieved by the formation of a large pit on the cell surface, which facilitates the transport of alginate to the periplasm, where it is captured by two binding proteins, AlgQ1 and AlgQ2, and delivered to an ABC transporter, consisting of two membrane-integral subunits, AlgM1 and AlgM2, and an ATPase, AlgS. Sequence analysis of the latter groups the transporter in the CUT1 family (Schneider, 2001). Both receptors bind alginate with Kd values around 0.1 μM. X-ray structures of AlgQ1 and AlgQ2, in their open form and in complex with an alginate tetrasaccharide, have been resolved and revealed a larger cleft in between the N- and the C-terminal lobes as observed for other solute receptors. Many positively charged residues in the cleft enable both proteins to bind preferentially to alginate (reviewed in Murata et al., 2008, original references therein).
Other sugars. In bacteria, glucose is often internalized as glucose-6-phosphate via the PTS. In the hyperthermoacidophile S. solfataricus, belonging to the phylum Archaea that apparently lack PTS, a glucose ABC transporter was identified. The SBP binds glucose with a Kd of 0.4 μM at low pH (Albers et al., 2004). Crystal structures of the NBD, GlcV, with bound nucleotide and in the ligand-free state, were solved (Verdon et al., 2003).
A transporter specific for maltose and trehalose (disaccharide of α-1,1-linked glucose residues, originating from plants and fungi) that also accepts sucrose and palatinose (Silva et al., 2005) was characterized in the archaeon T. litoralis (Xavier et al., 1996). The structure of the cognate-binding protein complexed with trehalose is known (Diez et al., 2001) and the biochemical properties of the complete transporter (Greller et al., 2001) and the isolated NBD (MalK) (Greller et al., 1999) were investigated. The crystal structure of MalK was resolved, but the conformation of the dimer is likely to be an artifact because it differs from almost all other solved NBD dimers (Diederichs et al., 2000).
A transporter, AglEFGK, allowing growth on a variety of α-glucosides, including sucrose, maltose and trehalose, was identified in the root–nodule symbiont Sinorhizobium meliloti (Willis & Walker, 1999). The sucrose isomer palatinose (6-O-α-d-glucopyranosyl-d-fructofuranose) is produced from sucrose by some bacteria as a means of carbon storage. The plant tumorigenic bacterium Agrobacterium tumefaciens utilizes palatinose as a carbon source, which is internalized by the PalEFGK transporter displaying homology to the putative maltose/trehalose transporter of S. meliloti. Trehalose, which is an osmoprotectant in S. meliloti, is not transported by the Pal system as demonstrated by analysis of a palK mutant. In addition, the mutation only affected growth on palatinose, but not on glucose, maltose, sucrose or galactose (de Costa et al., 2003).
Besides ABC transporters for the uptake of glucose, galactose and xylose, the gram-negative bacterium Agrobacterium radiobacter also possesses a binding protein-dependent transport system for lactose, LacEFGK, which is usually transported by pmf-driven porters or PTS (Greenwood et al., 1990). The system displays considerable sequence similarity to the MalEFGK transporter of E. coli, which is underscored by the observation that the ATPase subunit LacK can replace MalK in maltose transport (Wilken et al., 1997).
Ribose, a degradation product of nucleosides, is transported in E. coli by the RbsBCA system, the best-characterized member of the CUT2 family (Schneider, 2001).
The hyperthermophilic bacterium T. maritima has multiple archaeal homologs of ABC transporters from the PepT subfamily characterized as sugar transporters of various specificities (Nanavati et al., 2006). Among 15 SBP components of these ABC transporters tested for sugar binding using fluorescence spectroscopy, 11 were characterized as sugar transporters with their own specific profiles of substrate specificities. Among the substrates bound by these T. maritima SBP proteins were several monosaccharides (xylose, ribose), disaccharides (cellobiose, laminaribiose, xylobiose, mannobiose, maltose, trehalose), various oligosaccharides, as well as myo-inositol and α-1,4-digalacturonic acid.
Polyols. Sugar alcohols (polyols) are utilized by a variety of bacteria and often internalized by PTS. The existence of an ABC transporter with a specificity for d-mannitol and d-glucitol (sorbitol) and homology to the maltose ABC transporter was deduced from sequence analysis in the purple, nonsulfur bacterium Rhodobacter sphaeroides Si4 (Stein et al., 1997). An operon, mtlEFGK, encoding genes for a transporter with a similar specificity (mannitol, araitol, glucitol) was identified in Pseudomonas fluorescens (Brünker et al., 1998).
Erythritol is a likely carbon source of root–nodule bacteria from the species R. leguminosarum. An operon encoding an ABC transporter, EryABCD, was identified on a plasmid of R. leguminosarum bv. viciae, which is induced by erythritol. A mutant lacking an intact eryA gene that encodes the putative binding protein was impaired in its capability to compete for nodulation against the wild type (Yost et al., 2006). Orthologs were found in the genomes of Brucella sp..
γ-Hexachlorocyclohexane (γ-HCH). Sphingomonas japonicum strain UT26 can grow aerobically on γ-HCH, an insecticide, as the sole source of carbon and energy. The compound is degraded to β-ketoadipate (Lal et al., 2010). Analysis of mutants identified a gene cluster, linKLMN, encoding a putative ABC transporter to be involved in γ-HCH metabolism (Endo et al., 2007). While LinL is a typical nucleotide-binding protein, LinK has features of a membrane-integral subunit, but lacks the EAA motif of canonical ABC importers. LinM has an N-terminal periplasmic signal peptide and is similar to Mce proteins of Mycobacteria (see Cholesterol) and might thus be the SBP of the putative transporter. LinN has the features of a lipoprotein. Mutational analysis suggested to the authors that the LinKLMN transporter might contribute to a controlled access of the hydrophobic and thus a potentially toxic substrate to the membrane by slowing down γ-HCH diffusion into the cell (Endo et al., 2007).
Phthalate. The capability of bacteria from the genus Burkholderia to utilize phthalate as the sole source of carbon and energy by degradation via protocatechuate is well documented (Chang & Zylstra, 1998 and references therein). Phthalates, or phthalate esters, are mainly used in the chemical industry as plasticizers to soften polyvinyl chloride. Besides a secondary transporter, uptake is achieved by the action of an ABC transporter, OphFGH. The encoding genes are part of an operon, together with the gene for a specific outer membrane porin, OphP (Chang et al., 2009). The putative SBP, OphF, does not show high levels of similarity to other SBPs in the database. Phthalate uptake is strongly inhibited by 4-chlorophthalate and salicylate, suggesting that both compounds are accepted as substrates.
Cholesterol. In actinobacteria, including Mycobacterium tuberculosis and Rhodococcus jostii, evidence for a novel type of ABC transporter for the uptake of cholesterol that is used as a carbon source was provided. Mycobacterium tuberculosis, which is the causative agent of tuberculosis, requires host-derived cholesterol for persistence (Pandey & Sassetti, 2008). Genes encoding two ABC-type membrane-integral proteins are part of an operon (mce) containing between nine and 13 genes of an otherwise unknown function. The Mce1A protein is considered a candidate for a solute-binding lipoprotein of the system (Sutcliffe & Harrington, 2004). In fact, M. tuberculosis contains four mce operons and it was proposed (Pandey & Sassetti, 2008) that each putative ABC transporter is energized by an interaction with a genetically unlinked common nucleotide-binding protein of the Mkl family (Davidson et al., 2008). For the Mce4 system of R. jostii, cholesterol uptake was verified by transport assays (Mohn et al., 2008). Related operons were also identified by bioinformatics in gram-negative bacteria. Because mutations were found to affect cell envelope integrity, it was proposed that the substrates might be organic acid precursors of cell envelope biogenesis (Casali & Riley, 2007). How this proposal correlates with the experimental findings for cholesterol transport as cited above remains to be elucidated.
Bicarbonate. In the cyanobacterium Synechococcus sp., a gene cluster encoding a bicarbonate ABC transporter was identified by mutational analysis and uptake experiments that is activated under CO2-limiting growth conditions (Omata et al., 1999). The transporter comprises a solute-binding lipoprotein, CmpA, a membrane-integral subunit, CmpB, and two ATPase subunits, CmpC and CmpD. Binding experiments with recombinant CmpA demonstrated that HCO3− (Kd=0.5 μM) rather than CO2 is the substrate (Maeda et al., 2000). The proteins are homologous to the nitrate ABC transporter (NrtABCD) of Synechocystis sp. (Koropatkin et al., 2006; see Nitrogen sources). This is underscored in the crystal structure of CmpA, which was resolved in complex with bicarbonate and carbonic acid and in the absence of ligands. Bicarbonate was found to bind in a nearly identical position as nitrate in NrtA. Bicarbonate binding is accompanied by a Ca2+ ion that might act as a cofactor or as a cosubstrate in bicarbonate transport (Koropatkin et al., 2007b). Moreover, the ATPase subunit CmpC, like NrtC of the nitrate transporter, contains a C-terminal solute-binding domain involved in the regulation of the transporter's activity (see Nitrogen sources). Interestingly, the C-terminal domain is about 50% similar to NtrA, the SBP of the nitrate transporter, and it was proposed that nitrate and not bicarbonate is the likely substrate (Koropatkin et al., 2006).
Carbon and nitrogen sources
Peptides. Peptides can serve as sources of nitrogen or amino acids in auxotrophs such as lactic acid bacteria. Thus, ABC transporters mediating the uptake of peptides play an important role in the nutrition of these organisms. In addition, peptide transporters play crucial roles in signaling processes and in virulence (reviewed in Detmers et al., 2001; Doeven et al., 2005), which will be discussed in Oligopeptide transporters. Peptide transporters specific for di- and tripeptides (Dpp) or oligopeptides containing five and more residues) (Opp) are grouped within the PepT family. Generally, like the well-characterized systems of S. Typhimurium and L. lactis, oligopeptide transporters consist of an SBP, OppA, and a heterodimer each of the membrane-integral subunits, OppB and OppC, and the ATP-binding subunits, OppD and OppF (Detmers et al., 2001). The oligopeptide-binding proteins determine the selectivity of the system as was shown in an in vitro study using purified and membrane-reconstituted Opp of L. lactis (Doeven et al., 2004). Structures of DppA from E. coli and three OppA proteins from different organisms revealed that the specificity for peptides is determined by hydrogen bonds with the ligand backbone. The side chains are located in pockets that can accommodate any side chain, which is in line with the observed lack of sequence specificity for the ligands (reviewed in Doeven et al., 2005). Most OppA proteins bind peptides with two to seven residues. By contrast, the OppA protein of L. lactis MG 1363 handles peptides up to 35 residues (Doeven et al., 2005). Crystal structures of OppA in the open and closed (liganded) conformations provided a clue for this unusual property (Berntsson et al., 2009). The protein has an enlarged substrate-binding cavity due to the movement of two loops to the surface, which, in other OppA orthologs, confine the binding cleft. Analysis of peptides bound to OppA revealed a preference for peptides between nine and 17 residues, enriched in prolines. Crystal structures in complex with peptides revealed a hydrophobic pocket that accommodates one of the peptide's side chains, which was often an isoleucine. These findings coincide with the organism's requirement for branched-chain amino acids and its preference for proline-rich casein.
The diversity of oligopeptide ABC transporters in L. lactis and other lactic acid bacteria is reflected by the observation that most strains contain more than one copy of an Opp system and in some organisms more than one peptide-binding protein is associated with a given transporter (Lamarque et al., 2004; Doeven et al., 2005). Likewise, in the genome of Borrelia burgdorferi, the causative agent of Lyme disease, which is deficient in genes for the biosynthesis of amino acids, a single Opp transporter is encoded that might interact with five different peptide-binding proteins, OppA1–OppA5 (Wang et al., 2004). The expression of the encoding genes seems to be controlled by different transcription factors (Medrano et al., 2007). Similarly, four Opp systems were identified by in silico analysis in S. aureus and shown to be expressed differently (Hiron et al., 2007). However, only one system (Opp3) was required for the growth of S. aureus in a medium deficient in glutamate/glutamine, but supplemented with glutamate/glutamine-containing peptides (4-mers to 8-mers).
The OppBCDE transporter of gram-negative bacteria is involved in recycling of cell wall components that are delivered by the specific peptide-binding protein, MppA. The regular receptor, OppA, is not involved. Amidases release murein peptides, such as l-alanyl-γ-d-glutamyl-meso-diaminopimelate, into the periplasm, from which they diffuse out of the cell, or may enter the cytoplasm via the MppA-OppBCDF transporter (Park et al., 1998). MppA, which exhibits sequence similarity to OppA, was also reported to transport heme into E. coli cells when combined with the dipeptide ABC transporter, DppBCDF. However, the cognate-binding protein, DppA, could replace MppA (Létofféet al., 2006).
Amino acids. Prokaryotes can utilize amino acids as carbon and/or nitrogen sources or they are required as precursors under auxotrophic growth conditions. Transport systems with overlapping specificities belonging to the pmf-driven major facilitator superfamily and to the ABC transporter superfamily coexist in many organisms. ABC transporters mediating the uptake of amino acids are grouped into three families: the polar amino acid transporters (PAAT), the hydrophobic amino acid transporters (HAAT) and the methionine porters (MUT; discussed under Sources of sulfur) (Hosie & Poole, 2001; Zhang et al., 2003). The histidine-lysine/arginine/ornithine transporter of S. Typhimurium is the prototype of the PAAT family (reviewed in Hosie & Poole, 2001; Schneider, 2003). Genetic, molecular biological and biochemical studies on the system by G. Ames and colleagues contributed substantially to the current knowledge on ABC transporters. The transporter consists of two SBPs, HisJ and LAO (product of the argT gene), two transmembrane subunits, HisQ and HisM, each spanning the membrane only five times, and a homodimer of the ATPase subunit, HisP. HisJ and LAO exhibit high affinities in the nanomolar range for histidine and lysine/arginine/ornithine, respectively, but each binds the preferred substrate(s) of the other with about 10-fold lower affinities. The proteins are 70% identical and may have thus evolved by gene duplication. The crystal structures of HisJ and LAO in complex with their substrates revealed that the ligand preference of both proteins might be the result of a single amino acid exchange (Oh et al., 1994a, b). HisP was the first NBD of any ABC transporter whose structure was resolved, showing the typical nucleotide-binding (RecA-like) and α-helical subdomains (Hung et al., 1998). The reported HisP dimer structure, however, has the NBDs in a different orientation than the vast majority of all other NBD structures and is most likely an artifact. The complete transporter was purified and intensively characterized in proteoliposomes (Ames et al., 2001).
Other PAAT family members closely related to the histidine transporter that have been partially characterized include the E. coli glutamine (GlnH-PQ) and arginine-specific (ArtIJ-QMP) transporters (reviewed in Hosie & Poole, 2001) and the arginine/lysine/ornithine/histidine transporter (ArtJ-MP) of the thermophile G. stearothermophilus DSMZ 13240 (Fleischer et al., 2005; Vahedi-Faridi et al., 2008). The latter is homologous to the YqiXYZ porter identified in B. subtilis (Sekowska et al., 2001). Crystal structures of ArtP of G. stearothermophilus, which is 52% identical to HisP, in complex with nucleotides are known and show a dimer organization consistent with that of most other NBDs (PDB code 2OUK, 2OLK, 2OLJ, 2QOH, 3C4J and 3C41).
Two other close relatives of the histidine transporter, the OccT-QMP and NocT-QMP systems, are worth mentioning as they mediate the uptake of the arginine-derived modified amino acids octopine and nopaline as nutritional sources in the plant pathogen A. tumefaciens. Agrobacterium tumefaciens causes the formation of a tumorous growth on a wide variety of dicotyledonous plants and elicits the synthesis of a number of modified amino acids called opines by the plant. Both transporters were studied by uptake experiments with intact cells only (Zanker et al., 1992). Transporters for other opines are members of the PepT and POPT families, respectively (Table 1).
ABC transporters mediating the uptake of acidic amino acids were characterized from several bacteria. In Corynebacterium glutamicum, a high-affinity glutamate transporter, GluBCDA, was identified by mutational analysis and transport assays with intact cells. Whether the transporter is specific for glutamate or also accepts aspartate was not investigated (Kronemeyer et al., 1995). The AatJMQP transporter of Pseudomonas putida was shown to transport glutamate and aspartate. The purified SBP, AatJ, binds glutamate and asparagine with an equally high affinity (Kd values of 0.34 and 1.3 μM, respectively), while glutamine and asparagine are recognized with a much lower affinity (Singh & Rohm, 2008). In contrast, transport competition assays revealed that glutamate, aspartate, glutamine and asparagine are substrates of the BztABCD transporter from R. capsulatus (Zheng & Haselkorn, 1996).
An interesting ensemble of amino acid transporters was found in the filamentous cyanobacterium Anabaena sp. that can fix molecular nitrogen in differentiated cells termed heterocysts. Basically, only three ABC transporters are responsible for amino acid uptake of the organism. While the NatFGH transporter recognizes acidic and neutral amino acids, the BgtAB system takes up basic amino acids. In addition, the NatABCDE porter belonging to the HAAT family prefers proline and hydrophobic amino acids. The BgtAB system is composed of the ATPase subunit, BgtA, and BgtB, a fusion of an N-terminal solute-binding domain and a C-terminal TMD. The bgtA gene is not linked to bgtB, but included in the gene cluster encoding the NatFGH proteins and shared by both transporters as revealed from uptake assays. Both Nat systems appear to contribute to nitrogen fixation (Picossi et al., 2005; Pernil et al., 2008).
While most of the transporters are rather specific for chemically closely related amino acids, a few systems accepting a wide range of amino acids are known that seem to be widespread. The best-characterized general amino acid transporter is the AapJQMP porter of the root symbiont R. leguminosarum, which prefers basic and acidic amino acids, but also transports aliphatic amino acids (reviewed in Hosie & Poole, 2001). Transport experiments with intact cells revealed that the Aap system, the branched-chain amino acid transporter, Bra, of R. leguminosarum, and the histidine transporter of S. Typhimurium mediate not only the uptake but also the export of amino acids, which might be a means to cope with the accumulation of amino acids as a consequence of metabolism. This finding challenges the concept of the unidirectional transport of ABC import systems depending on an extracellular binding protein (reviewed in Hosie & Poole, 2001). The authors assumed that substrate molecules must gain access to a (low affinity) binding site within a transporter from the cytoplasmic site that does not necessarily have to be identical to the one used for uptake. How this could be achieved was not yet further explored under in vitro conditions and whether it is only an intrinsic property of amino acid transporters is not known. The currently available crystal structures do not provide a clue in favor of this notion.
Within the HAAT family, transporters for branched-chain amino acids have been characterized from E. coli, S. Typhimurium and Pseudomonas aeruginosa (reviewed in Hosie & Poole, 2001). The LIV-1 system of E. coli consists of two SBPs, LivJ and LivK, and a membrane-bound complex comprising two membrane-integral subunits, LivH and LivM, and two ATPase subunits, LivG and LivF. Crystal structures of the LivG homolog from Methanococcus janaschii in its monomeric form have been resolved (Karpowich et al., 2001). LivK was originally reported to be specific for leucine (Kd∼1 μM), while LivJ binds leucine, isoleucine and valine with similar affinities (Kd∼0.1–1 μM) and threonine, serine and alanine with a lower affinity. In a more recent study, using an E. coli strain deficient of all known transporters for aromatic amino acids, it was demonstrated that the LIV-1 system also transports phenylalanine. Uptake studies further revealed that the transporter, when equipped with LivK, accepts isoleucine and valine similar to leucine (Koyanagi et al., 2004).
A similar modular organization is found for the Salmonella LIV-1 transporter, whereas the homologous system from P. aeruginosa (BraC-DEFG) contains only one SBP (BraC). The Bra system of P. aeruginosa is one of the few that have been characterized in proteoliposomes, thereby demonstrating that alanine and threonine are true substrates (Hoshino et al., 1992). Other members of the HAAT family display a much broader range of substrate specificity (Hosie & Poole, 2001).
Both broad-specificity amino acid transporters of R. leguminosarum (Aap and Bra) are required for effective nitrogen fixation in pea nodules (Lodwig et al., 2003). It was shown that bacteroids (differentiated forms of the bacteria within root nodules) become symbiotic auxotrophs for branched-chain amino acids, and depend on the plant for supply (Prell et al., 2009).
γ-Aminobutyric acid (GABA) was suggested as a candidate amino acid (beside glutamate) that, in root–nodule symbiosis, is donated by the plant to the bacteroid in exchange for ammonia as a result of nitrogen fixation (Lodwig et al., 2003). In mutants of R. leguminosarum bv. viciae 3841 that grow faster than the wild type on GABA as the sole source of carbon and nitrogen, an ABC transport system, GtsABCD, exhibiting specificity for GABA and related compounds was identified (White et al., 2009). The transporter, belonging to the POPT family (Table 1), consists of an SBP, GtsA, two transmembrane subunits, GtsBC, and an ATPase, GtsD. The Gts system was not expressed in pea bacteroids, thereby questioning a role in amino acid cycling between host and symbiont. Rather, the authors consider it likely that this function is achieved by the Bra system, which was also shown to transport GABA (Hosie et al., 2002).
Nucleosides. Uptake or scavenging of ribonucleosides from the environment is a means to provide cells with precursors for nucleic acid synthesis and sugars as a source of carbon and energy. In S. mutans, the predominant causative agent of dental caries, an ABC transporter for the uptake of ribonucleosides, was identified as the only member of the carbohydrate uptake family CUT2 present in this organism (Webb & Hosie, 2006). The transporter, RnsBACD, consists of an ATPase subunit, RnsA, two membrane-integral subunits, RnsCD, and a solute-binding lipoprotein, RnsB. RnsA, like RbsA and other ATPases of the CUT2 family, is a fused heterodimeric protein, containing two putative NBDs. Uptake experiments with intact cells using radiolabeled cytidine in combination with competing solutes revealed the acceptance of most ribonucleosides, whereas ribose and nucleobases were not recognized.
An ortholog of the Rns system was found in the genome of Treponema pallidum, the causative agent of syphilis (Deka et al., 2006). The putative transporter, PnrABCDE, which could not be studied in intact cells due to the inability to cultivate the organism in vitro, was characterized via its cognate-binding protein, PnrA. The protein, which is a lipoprotein despite the dual membrane system of the cell envelope, was demonstrated by isothermal titration calorimetry to bind purine nucleosides with a Kd of ∼0.1 μM. The crystal structure of PnrA complexed with inosine (likely procured from the E. coli host) was resolved and revealed a polypeptide with structural similarities to family 1-binding proteins, such as ribose- and glucose/galactose-binding proteins of E. coli. Because T. pallidum lacks the capacity for de novo synthesis of purines, the transporter might enable the organism to take up nucleic acid precursors from the human host.
Choline, glycine betaine, proline betaine. Quaternary ammonium compounds are commonly used as osmoprotectants in many bacteria and archaea (Welsh, 2000). In a few bacteria, including the root–nodule symbiont S. meliloti, osmoprotectants can be utilized at low osmolarity exclusively as carbon and nitrogen sources or in addition to osmoprotection. Choline (trimethylammonium) is a main constituent of eukaryotic plasma membranes in the form of phosphatidylcholine and is thus readily available in different environments including the soil and the rhizosphere. For its use as a nutrient, it has to be enzymatically converted to glycine betaine, from which pyruvate is subsequently formed (Smith et al., 1988). Uptake of choline in S. meliloti is achieved by an ABC transporter composed of the SBP, ChoX, the membrane-integral subunit, ChoW, and the ATP-binding subunit, ChoV. The proteins share the highest similarities to the corresponding subunits from ProU-like transporters involved in glycine betaine and proline uptake for osmoprotection (Dupont et al., 2004). However, the transporter is induced by choline, but not by high salt. The purified ChoX protein binds choline with a high affinity (Kd, 2.7 μM) and acetylcholine with a low affinity, but not glycine betaine or proline betaine. The crystal structures of ChoX, in complex with its ligands and in an unliganded, closed form, were determined (Oswald et al., 2008). Most recently, a crystal structure of ChoX in a semi-open substrate-free form was reported that was attributed to the movement of a subdomain in the N-lobe that might be present in other SBPs as well (Oswald et al., 2009).
In P. aeruginosa and Pseudomonas syringiae, the CbcXWV system mediates the uptake of choline, betaine and carnitine. The choline-binding protein, CbcX, is an ortholog of ChoX of S. meliloti. Two other binding proteins, BetX and CaiX, with specificities for betaine and carnitine, respectively, that are genetically unlinked, deliver their substrates to the CbcWV transporter. Interestingly, transport experiments revealed that CheX and BetX compete with CaiX-betaine for docking to the core transporter in their liganded forms only, thereby suggesting that productive receptor–transporter interactions are dependent on the presence of a substrate (Chen et al., 2009).
Two ABC transporters have been implicated in the transport of proline betaine (N,N-dimethylproline) in S. meliloti. The Hut transporter displays a high affinity for histidine, proline and proline betaine and is controlled at the transcriptional level by histidine, but not by salt stress. This is different from the ProU system of E. coli with which Hut shares homology, but is consistent with a role of the transporter in catabolism (see also Uptake of compatible solutes). In contrast, the Prb system, with a periplasmic-binding protein, PrbA, two membrane-integral subunits, PrbB and PrbC, and an ATP-binding subunit, PrbD, is a member of the oligopeptide transporter family (Alloing et al., 2006). prb gene expression is induced by both proline betaine and sodium chloride, thereby differing from the cho genes. Moreover, uptake experiments with intact cells revealed that besides proline betaine, glycine betaine and choline are substrates.
EDTA. Bacterium BNC1, closely related to Mesorhizobium and Agrobacterium sp., was demonstrated to degrade the metal-chelating agent EDTA for use as a nitrogen source. Uptake of EDTA is probably mediated by the EppABCD transporter, whose encoding genes are adjacent to the gene for EDTA monooxygenase. Purified SBP, EppA, binds free EDTA with a Kd of 25 nM, but not stable metal–EDTA complexes (Zhang et al., 2007).
Nitrate, nitrite, cyanate, urea. Besides amino acids, prokaryotes can utilize a wide range of nitrogen sources that are transported by specific ABC uptake systems belonging to the NitT family.
Cyanobacteria require nitrate for growth, which is usually severely limited in aquatic environments. Organisms such as Synechococcus sp. and others are equipped with a high-affinity ABC transporter for the uptake of nitrate and nitrite. The Nrt system consists of a solute-binding lipoprotein, NrtA, localized to the outer leaflet of the inner membrane, which is an unusual feature of binding proteins from gram-negative bacteria, but typical for cyanobacteria, a membrane integral subunit, NrtB, a canonical ATPase subunit, NrtD, and a unique NBD-SBP fusion protein, NrtC (Omata, 1995; Maeda & Omata, 1997). As already mentioned (Uptake of compatible solutes), the Nrt system shows significant similarities to the CmpA-BCD transporter, mediating the uptake of bicarbonate in cyanobacteria (Omata et al., 1999). NrtA has a signal sequence containing a double arginine motif at its N-terminus, which marks it as a substrate for the Tat export system. The crystal structure of NrtA was resolved in complex with nitrate (Koropatkin et al., 2006) and revealed an α/β protein composed of two domains that encompass the binding cleft that belongs to family 2 of SBPs (Wilkinson & Verschueren, 2003). The nitrate flux through the transporter is inhibited by ammonium ions that bind to the C-terminal solute-binding domain of NrtC (Kobayashi et al., 1997).
The N-terminal domain of NtrC from Phormidium laminosum, when expressed separately, was found to bind ATP, but no ATPase activity could be detected (Nagore et al., 2003).
A transporter displaying sequence similarity to the Nrt (and Cmp) system, NasFED, was reported for the enterobacterium K. oxytoca, which can utilize nitrate and nitrite as nitrogen sources (Wu & Stewart, 1998). Here, the periplasmic-binding protein, NasF, lacks an N-terminal cysteine residue required for modification with fatty acids and thus, as expected for gram-negative bacteria, is not a lipoprotein. NasE is homologous to NrtB and NasD is about 47% identical to NrtD. No NrtC-like component was found, although nitrate uptake by the NasFED transporter was inhibited by ammonium.
In the cyanobacterium Synechococcus elongatus, an ABC transporter, previously identified as a cyanate porter, CynABD (Espie et al., 2007), was also shown to transport nitrite, in addition to the Nrt system (Maeda & Omata, 2009). CynA displays a high degree of sequence similarity to the periplasmic SBPs NrtA and CmpA. The transporter is thought to play a role in utilizing low concentrations of cyanate under nitrogen-limiting conditions and to allow those cyanobacteria that lack an Nrt system to assimilate nitrite.
Urea is a readily available nitrogen source due to its excretion by a variety of organisms in the environment. It can be metabolized by many microorganisms. An ABC transporter mediating the uptake of short-chain amides and urea (FmdDEF) was found in Methylophylus methylotrophus (Mills et al., 1998) and characterized in more detail in cyanobacteria (Valladares et al., 2002). The UrtABCDE porter of Anabaena sp. PCC 7120 consists of the periplasmic-binding protein, UrtA, two transmembrane subunits, UrtB and UrtC, and two ATP-binding subunits, UrtD and UrtE. The proteins share the highest sequence identities with the components of the branched chain amino acid transporter from P. aeruginosa. Uptake experiments with intact cells revealed a Km of about 0.11 μM. Expression of the genes was induced under nitrogen-limiting conditions. A similar system was identified in C. glutamicum, whose expression is under the control of the global nitrogen regulator of the organism, AmtR (Beckers et al., 2004).
Sources of sulfur
Prokaryotes obtain sulfur either from inorganic sulfate or from organosulfur compounds including sulfonates, sulfate esters, sulfur-containing amino acids and glutathione (Kertesz, 2001).
Sulfate/thiosulfate/taurine. Sulfate/thiosulfate ABC transporters are members of the SulT family. In E. coli, the transporter consists of two binding proteins with preferred specificities for sulfate (SuBP) and thiosulfate (CysP), respectively, two membrane components, CysT and CysW, and the ATPase, CysA. The crystal structures of SuBP of S. Typhimurium and CysA from Alicyclobacillus acidocaldarius have been resolved. In SuBP, sulfate is bound only by hydrogen bonds to neighboring amino acids and entirely dehydrated (Pflugrath & Quiocho, 1985, 1988). CysA revealed a C-terminal extension similar to those observed in the ABC proteins from CUT1 family members, but with an as yet unknown function (Scheffel et al., 2005). The cysPTWA genes, together with other cys genes required for the synthesis of cysteine, are repressed in the presence of cysteine. Expression is activated by action of the positive regulator, CysB, and the coinducer, N-acetylserine (van der Ploeg et al., 2001).
The utilization of taurine (2-aminoethanesulfonic acid) and alkanesulfates in E. coli and other bacteria requires the ABC transporters TauABC and SsuABC, belonging to the TauT family (Kertesz, 2001). Expression of the encoding genes is derepressed under conditions of sulfate or cysteine starvation (van der Ploeg et al., 2001). Analysis of deletion mutants revealed that components of both transport systems are not functionally interchangeable (Eichhorn et al., 2000).
Cysteine/cystine/methionine. In E. coli and S. Typhimurium, experiments with intact cells revealed the participation of two binding protein-dependent transport systems in the uptake of cystine. The FliY/YecS/YecC system, one of the E. coli porters, has a rather broad substrate specificity also recognizing diaminopimelic acid. None of the systems has been characterized further, which also holds for cysteine transporters (Hosie & Poole, 2001).
A cysteine-binding protein (CjaA) was purified and crystallized from Campylobacter jejuni, a food-borne pathogen and a leading cause of acute human gastroenteritis. CjaA, which is a conserved immunodominant protein in C. jejuni, was shown to specifically bind l-cysteine with a Kd of ∼0.1 μM, while the affinity for l-serine was >200-fold lower. Binding of other amino acids could not be detected (Müller et al., 2005).
In B. subtilis, two ABC transporters were identified by mutational analysis and uptake experiments with intact cells to mediate l-cystine transport (Burguière et al., 2004): TcyJKLMN and TcyABC. The former includes two SBPs, TcyJ and TcyK, exhibiting 57% identity with unknown specificities. However, besides cystine, the system accepts cystathionine, djenkolic acid and S-methyl-cysteine, while the TcyABC porter seems to be more specific for cystine. Expression of the genes encoding the TcyJKLMN system was high in the presence of methionine, but reduced in the presence of sulfate or cystine (Even et al., 2006). Expression of the TcyABC-encoding genes was low under all the conditions tested. TcyABC-like transporters are found in many gram-positive bacteria. The putative YxeMNO transporter that is most similar to the E. coli FliY/YecS/YecC is not involved in cystine uptake (Burguière et al., 2004). The first crystal structure of a cystine-binding protein, NGO372 from Neisseria gonorrhoeae, which displays 50% and 41% sequence identity, respectively, to TcyA of B. subtilis (Burguière et al., 2004) and BspA of Lactobacillus fermentum (Turner et al., 1999), was recently resolved (H. Bulut, F. Scheffel, S. Moniot, W. Saenger & E. Schneider, unpublished data).
ABC transporters that transport l- and d-methionine, formyl-l-methionine and likely organic sulfur compounds in gram-positive bacteria are clustered in the MUT family (Zhang et al., 2003). In E. coli, the long-known metD locus was shown to encode the MetNIQ transporter (Gál et al., 2002; Merlin et al., 2002) with the ATPase subunit, MetN, the membrane subunit, MetI, and the receptor, MetQ. These proteins were formerly known as ABC, YaeE and YaeC, respectively. Interestingly, the receptor, MetQ, is proposed to be a lipoprotein. The crystal structure of the Met(NI)2 complex at a 3.7 Å resolution was resolved and revealed an explanation for an allosteric regulatory mechanism that operates at the level of transport activity (Kadaba et al., 2008). Increased cellular concentrations of the transported ligand stabilize an inward-facing conformation of the transporter by binding to a site located in between the C-terminal extensions of the respective NBD dimers. As a consequence, the transporter is locked in an inactive state, thereby preventing further ligand uptake (Kadaba et al., 2008). A similar mechanism was found for the molybdate/tungstate transporter of Methanosarcina acetivorans, Mod(AB)2 (see Trace elements).
The MetNPQ transporter of B. subtilis, which is also distributed among other gram-positive bacteria, was shown to also transport methionine sulfoxide (Hullo et al., 2004). In S. mutans, the homologous AtmBDE system is involved in the uptake of l- and d-methionine, selenomethionine and homocysteine (Sperandio et al., 2007).
A methionine-binding lipoprotein (TP32) was identified from its crystal structure in T. pallidum. The protein shares significant similarity to the putative methionine-binding protein MetQ (YaeC) of E. coli and is thus suggested to be a constituent of a methionine ABC transporter (Deka et al., 2004). The crystal structure of a related protein, GNA1946, from Neisseria meningitidis, which is assumed to be a lipoprotein, also revealed l-methionine as the most likely substrate (Yang et al., 2009b).
Glutathione. Mutational analysis and transport assays revealed a novel type of ABC transporter in E. coli, mediating the uptake of glutathione that is used as the sole source of sulfur (Suzuki et al., 2005). The transporter is a member of the PepT family and consists of the solute receptor, YliB, two inner membrane components, YliC and YliD, and an ATP-binding subunit, YliA. Homologs are found in other enterobacteria.
Iron has a vital function for almost all prokaryotes due to its role in electron transfer proteins, for example cytochromes, and in some proteins, such as ribonucleotide reductase and soluble methane monooxygenase. Although iron is one of the most abundant elements on earth, its requisition is a major challenge for many prokaryotes because it is not readily available under most growth conditions. In the presence of oxygen and at neutral pH, iron forms insoluble hydroxides and most bacteria and certain fungi are forced to synthesize and secrete low-molecular-weight chelators (siderophores) that sequester Fe3+-ions with a high affinity. Only under anaerobic conditions and at pH values below 3 can ferric ions be transported without chelators (see Crosa et al., 2004 for review). Sufficient iron supply was also shown to contribute to the virulence of many pathogens. Consequently, iron transporters of pathogenic bacteria are virulence factors and are discussed in Uptake of nutrients from the host. Here, we will summarize the current knowledge on iron ABC transporters of E. coli K-12 and other nonpathogens.
Escherichia coli is equipped with three ABC transporters mediating the uptake of Fe3+-siderophores, namely, Fe3+-hydroxamate (FhuBCD), Fe3+-enterobactin (FepBCDG) and Fe3+-dicitrate (FecBCDE) (Köster, 2001). They are grouped within the FeCT family. While mutational and biochemical analyses have been performed for individual components of the transporters, an in vitro characterization of the assembled transport complexes has not been reported as yet. All three systems have in common the dependence of a specific outer membrane receptor, which, powered by the proton-motive force, translocates the substrate into the periplasm. Removal of a so-called plug, a domain of the polypeptide blocking the channel formed of β-strands (β-barrels), was suggested to be energy-consuming (Braun et al., 2004; Ferguson & Deisenhofer, 2004). The molecular basis of this process, which requires the presence of a cytoplasmic membrane protein complex comprising TonB, ExbB, and ExbD remains elusive (Postle & Larsen, 2007).
The purified Fe3+-hydroxamate-binding protein, FhuD, binds its substrates (ferric coprogen, ferric aerobactin, ferrichrome and albomycin) with dissociation constants in the low μM range (Braun et al., 2004). The crystal structures of FhuD that have been resolved in complex with several substrates place the protein into family 3 of solute receptors (Krewulak et al., 2004). The transmembrane subunit, FhuB, has about double the molecular mass of a typical TMD, each half spanning the membrane 10 times, and is proposed to represent a fusion of two TMDs with internal sequence homology. It was demonstrated that separate expression of the N- and C-terminal halves resulted in an active protein, thereby corroborating the above notion. The FhuC protein is a typical ABC ATPase (reviewed in Köster, 2001).
Fe3+-dicitrate is passing the outer membrane by the action of the FecA receptor. It is assumed that in the periplasm only Fe3+ is delivered to the binding protein, FecB, based on the finding that 10 times more radiolabeled Fe3+ is transported into the cytoplasm than citrate (Braun et al., 2004). The membrane-bound transport complex is composed of the transmembrane subunits FecB and FecC and the ATPase, FecE. The interaction of FecB with FecCD, as suggested for other ABC importers to initiate the transport cycle, was proposed based on the BtuF-CD structure to involve the formation of salt bridges. Evidence for this notion was presented recently by mutational analysis (Braun & Herrmann, 2007). The Fec system is induced by ferric citrate in the periplasm through a signaling pathway comprising the transmembrane protein FecR (Härle et al., 1995).
The first crystal structure of a binding protein in complex with a native catecholate siderophore was reported recently. The FeuA protein of B. subtilis binds its substrate, ferribacillibactin, by electrostatic interactions through two lysine and one arginine residue (basic triad) from the N- and C-terminal lobes, respectively, and via hydrogen bonds from two glutamine residues (Peuckert et al., 2009). A basic triad was also found in the structure of the enterobactin-binding protein CeuE from C. jejuni, which was crystallized in complex with an artificial substrate (Müller et al., 2006). FeuA belongs to family 3 solute receptors and is a component of the FeuABC-YusV transporter, which also imports ferrienterobactin (Ollinger et al., 2006).
Bacillus subtilis can also utilize the exogenous siderophore petrobactin, which, among others, is produced by members of the Bacillus cereus group, including the pathogen B. anthracis. Fe-petrobactin is internalized by the YclNOPQ transporter, comprising two transmembrane subunits, YclON, an ATPase, YclP and a solute-binding protein, YclQ. The crystal structure of YclQ revealed CeuE from C. jejuni as its closest relative, but also a similarity in shape to FeuA. The importance of three basic residues for interaction with the substrate was confirmed (Zawadzka et al., 2009).
Cyanobacteria have a high demand for iron to sustain photosynthesis. In Synechocystis PCC 6803, an iron transport system operates consisting of two SBPs, FutA1 and FutA2, which are 52% identical, a membrane-integral subunit, FutB, and an ATPase, FutC (Katoh et al., 2001). FutA1 and FutA2 bind iron directly without siderophores or anions, respectively, as revealed from their crystal structures. Iron is coordinated by four tyrosine and one histidine residue, as also observed in the crystal structure of the homologous FbpA protein of C. jejuni (Tom-Yew et al., 2005). Whether ferrous or ferric irons are the preferred ligands under physiological conditions is controversially discussed (Koropatkin et al., 2007a; Badarau et al., 2008). Moreover, the function of FutA1 as a periplasmic iron-binding protein has been questioned. Instead, it was suggested to act intracellularly under conditions of iron limitations. Accordingly, only FutA2, which has an export signal targeting it to the Tat secretion machinery, is proposed to deliver iron to the FutBC transporter (Badarau et al., 2008).
Manganese, zinc. Manganese plays a crucial role in the water-splitting enzyme associated with photosystem II of oxygen-evolving phototrophic bacteria and is an important metal for an oxidative stress response (Horsburgh et al., 2002). Zinc is not involved in electron transfer reactions due to its single oxidation state in solution. Rather, it is a cofactor in a variety of enzymes with diverse functions such as alkaline phosphatase, RNA polymerase, zinc finger proteins and some ribosomal proteins. Both transition metals (like iron) are toxic at higher intracellular concentrations and thus metal homeostasis pathways must operate. Among others, high-affinity ABC transporters for the uptake of these metals are part of such pathways. They cluster in the MCT family of the TC system and, based on sequence alignments, their cognate extracellular SBPs constitute cluster 9 of solute receptors (Claverys, 2001). Some have been recognized as important virulence factors of pathogens and are addressed in Uptake of nutrients from the host.
In the cyanobacterium Synechocystis sp. PCC 6803, a manganese ABC transporter, MntCAB, operates with a high affinity (Km, 1–3 μM), which is induced under manganese-starvation conditions (Bartsevich & Pakrasi, 1996). The crystal structure of its SBP, MntC, was resolved with bound Mn2+ and revealed the presence of an unusual, but functionally crucial disulfide (Rukhman et al., 2005).
Manganese ABC transporters related to an oxidative stress response were found in numerous bacteria (Claverys, 2001; Horsburgh et al., 2002). The putative iron/manganese transporter of the root–nodule symbiont S. meliloti was shown to be important for the organism's response to oxidative stress. The SitABCD system for which a homolog exists in S. Typhimurium (Kehres et al., 2002) (Uptake of nutrients from the host) favors manganese over iron as a substrate. A mutant lacking the solute-binding subunit, SitA, is symbiotically defective and displays elevated sensitivity to superoxide due to decreased levels of superoxide dismutase B. The S. meliloti SodB (also called SodA in older references) can operate with iron or manganese, therefore termed ‘cambialistic’, but exhibits a higher activity with the latter. The mutant was rescued by exogenous addition of 10 μM MnSO4, but not FeSO4, although the latter was not applied under reducing conditions to maintain the Fe(II) redox state. The authors concluded that the SitABCD transporter plays a crucial role in manganese uptake (Davies & Walker, 2007), which is consistent with data obtained for the Salmonella Sit system.
Escherichia coli and others have a ZnuABC system, consisting of the extracellular binding protein, ZnuA, a membrane-integral subunit, ZnuB, and an ATPase, ZnuC. Expression of the znuABC gene cluster is regulated by zinc and a specific repressor, Zur (Hantke, 2005). Crystal structures of Ec-ZnuA are available and show that both β-/α-domains are connected by an α-helix as it is characteristic of family 3 receptors (Wilkinson & Verschueren, 2003). Three conserved histidine residues and a glutamate were found to coordinate the zinc ion (Chandra et al., 2007; Li & Jogl, 2007; Yatsunyk et al., 2008). The glutamate was replaced by water in a structure of the homologous ZnuA protein from Synechocystis (Banerjee et al., 2003). Similar to MntC, the Ec-ZnuA structures show an unusual disulfide bond in the C-terminal domain, which might be important for structural integrity or regulation of Zn2+ binding. In one structure, a second metal-binding site with an unclear function was observed (Yatsunyk et al., 2008). Ec-ZnuA binds Zn2+ with an estimated Kd of <20 nM and other divalent cations, but not manganese. Only Zn2+, Cd2+ and Cu2+ caused conformational changes in the protein thought to be required for metal delivery to the cognate transport complex (Yatsunyk et al., 2008). A highly charged and mobile loop observed in the vicinity of the binding cleft is proposed to act as a zinc chaperone to facilitate acquisition (Banerjee et al., 2003).
The crystal structures of two other proteins, PsaA from S. pneumoniae and TroA from T. pallidum, were resolved in complex with Zn2+, but the nature of the physiological substrate (zinc or manganese) is still controversially discussed (Hantke, 2005). A binding study with TroA purified from E. coli revealed that Zn2+, Mn2+ and possibly iron might be substrates, but that the transcriptional regulator of the tro operon, TroR, is likely to bind Zn2+ (Hazlett et al., 2003).
Nickel. In E. coli, Ni2+ is an essential cofactor for NiFe-hydrogenases that operate only under anaerobic conditions. To fulfill the demand for Ni2+ ions, the nikABCDE operon encoding an ABC transporter is expressed in close correlation with the hydrogenase expression levels. Positive regulation of nikABCDE is achieved by the redox sensor FNR while transcription is repressed in response to excess Ni2+ by NikR (reviewed in Eitinger & Mandrand-Berthelot, 2000). Furthermore, nikABCDE expression is also under the control of the nitrate-regulatory system (Rowe et al., 2005). The transporter consists of two heterodimers representing the transmembrane (NikBC) and nucleotide-binding subunits (NikDE), and of a SBP, NikA. Because of sequence similarities to oligopeptide transporters, it is grouped within the PepT family of the TC system. NikA was purified and a Kd for Ni2+ was estimated to be around 0.1 μM based on equilibrium dialysis and quenching of intrinsic tryptophan fluorescence, while that for Co2+ was 10-fold higher (de Pina et al., 1995). In a subsequent study, using isothermal titration calorimetry, unphysiologically high Kd values of ∼10 and 250 μM were reported for Ni2+ and Co2+, respectively (Heddle et al., 2003). The lower affinity for Ni2+ found by this group corroborates with a study using fluorescently labeled NikA (Salins et al., 2002). Although several crystal structures of NikA have thus far been reported, a clear-cut answer on how Ni2+ is coordinated in the protein remains elusive. Heddle et al. (2003) presented structures obtained in the absence and presence of Ni2+. In the latter, the ligand is not occluded in the binding cleft, but is rather accessible to the solvent, which is in contrast to other binding proteins. Also, the rotational motion between both lobes upon binding Ni2+ was less than that observed for related binding proteins. In 2005, Cherrier et al. (2005) reported on a refined structure (1.8 Å resolution) of NikA in complex with FeEDTA(H2O)−. These authors, on close inspection of the data deposited by Heddle et al. (2003), arrived at the conclusion that the nature of the ion in the ‘Heddle’ structure remains unclear and could very well be an iron. Among other considerations, this was based on the fact that both groups used EDTA-containing buffers for the purification of NikA. Moreover, it was speculated that Ni2+ binding by NikA might require a chelating cofactor. This notion was further corroborated by the same group by resolving the crystal structure of NikA prepared in the absence of EDTA (Cherrier et al., 2008). Here, an unidentified chelator that might be butane-1,2,4-tricarboxylate was found to contribute to the binding of a transition metal ion of unknown identity. To make the picture even more complex, NikA was also shown to bind heme under anaerobic growth conditions, although the binding site is predicted to be remote from the nickel-binding cleft (Shepherd et al., 2007).
Homologs of the NikABCDE system with an experimentally confirmed function in nickel uptake were reported for Brucella suis (Jubier-Maurin et al., 2001) and Yersinia pseudotuberculosis (Sebbane et al., 2002). Because of the similarity among the SBPs within the PepT family, it is difficult to divide its members by amino-acid sequence comparisons into subclasses with substrate specificity for either metal ions or peptides. Based on genomic colocalization with genes for nickel-dependent enzymes or the presence of upstream NikR-binding sites, a subset of the PepT family was identified as metal transporters. A function in Ni2+ uptake has been ascribed to the vast majority of the members of this subset (Rodionov et al., 2006; Zhang et al., 2009). (see Uptake of Ni2+ and Co2+ ions, additional aspects of Ni2+ and Co2+ uptake).
Copper. Methanobactin, a siderophore-like molecule with a peptidic nature, is implicated in copper uptake in methanotrophic methylotrophs. Cu2+ is an essential cofactor of particulate methane monooxygenase. The acquisition of copper ions is achieved by methanobactin. Although nothing is currently known on the nature of the transporters involved, a TonB-dependent outer-membrane transporter and an ABC transporter in the cytoplasmic membrane were predicted for internalization of the Cu–methanobactin complex (Balasubramanian & Rosenzweig, 2008).
Molybdate/tungstate/vanadate. Molybdenum serves as a cofactor in a variety of enzymes, including (1) nitrogenase of nitrogen-fixing bacteria such as cyanobacteria or root–nodule symbionts and (2) a large group of pterin-based molybdenum enzymes in all kingdoms of life that are grouped into three families represented by sulfite oxidase, xanthine oxidase and dimethyl sulfoxide reductase (DMSOR). The DMSOR family includes many more enzymes involved in electron-transport chains under anoxic conditions, such as nitrate reductase and formate dehydrogenase (see Zhang & Gladyshev, 2008; Schwarz et al., 2009 for recent reviews). Molybdenum is never directly bound by an enzyme, but always via a cofactor, the ‘FeMoco’ (nitrogenase only) or a molybdenum cofactor (Moco), whose structure is derived from a metal-binding pterin (MPT). The same holds true for tungsten, which is found as a relative of Moco (called Wco) in tungsten-containing formate dehydrogenase (a member of the DMSOR family) and Wco-containing aldehyde : ferredoxin oxidoreductase (see Andreesen & Makdessi, 2008 for a review).
The E. coli ModABC transporter is the best-characterized uptake system for the naturally predominant form of Mo, the anoxyion molybdate (Self et al., 2001). It consists of the binding protein, ModA, the transmembrane subunit, ModB, and the ATPase, ModC. ModA binds molybdate with a very high affinity in the nanomolar range and, at equimolar quantities, also tungstate. Crystal structures of ModA in complex with molybdate and tungstate are available (Hu et al., 1997). Expression of the modABC genes is tightly regulated by the repressor, ModE, and requires molybdate starvation. Interestingly, the E. coli sulfate ABC transporter mediates molybdate uptake with a low affinity while, likewise with a low affinity, the ModABC system accepts sulfate as a substrate (Kertesz, 2001; Self et al., 2001).
The crystal structure of a molybdate/tungstate transporter, ModBC, from the sulfate-reducing archaeon Archaeoglobus fulgidus in complex with its cognate-binding protein, ModA/WtpA, has been resolved (Hollenstein et al., 2007). In contrast to ModA proteins from E. coli and Azotobacter vinelandii, which bind molybdate and tungstate as a tetrahedral complex, the crystal structures of ModA/WtpA of A. fulgidus and four other archaebacterial molybdate/tungstate-binding proteins revealed an octahedrally coordinated central metal ion (Hollenstein et al., 2009).
The crystal structure of a homolog from M. acetivorans, ModBC, revealed a C-terminal extension of the ATPase subunit, ModC, which provides the basis for the mechanism of trans-inhibition of transport similar to the methionine transporter of E. coli (Gerber et al., 2008) (Sources of sulfur). These regulatory domains provide two anoxy-binding pockets for molybdate/tungstate. When occupied by a substrate, the transporter is locked in the ‘inward-facing’ conformation, resulting in an inactive ATPase activity. Transport activity i.e. energy costs, can thus be controlled by cellular levels of the substrate.
A clear preference for tungstate over molybdate is exhibited by the receptor TupA of the TupABC transporter from the amino acid-degrading bacterium Eubacterium acidaminophilum (Makdessi et al., 2001). Unpublished data obtained by isothermal titration calorimetry revealed a Kd for tungstate of 0.2–1 nM, at least 1000-fold lower than that for molybdate. The crystal structure of TupA in the unliganded form was resolved (reported in Andreesen & Makdessi, 2008).
The genome of C. jejuni, which causes gastrointestinal infections, contains two distinct ABC transport systems (Cj0300–0303 and Cj1538–1540) with similarity to molybdate transporters and the tungstate transporter of E. acidaminophilum, respectively (Smart et al., 2009). Binding studies with the purified SBPs using isothermal titration calorimetry and intrinsic tryptophan fluorescence revealed equal affinities of Cj0303 (ModA) for molybdate and tungstate (Kd∼4–8 nM), but a clear preference of Cj1540 (TupA) for tungstate over molybdate (5 × 104-fold). Tungstate is bound with an unusually low Kd of 1 pM. By mutational analysis, it was demonstrated that a tupA strain displays a significant reduction in formate dehydrogenase activity, suggesting a role of tungstate as a cofactor of this enzyme (Smart et al., 2009). These data were corroborated by a study of Taveirne et al. (2009), who monitored the activities of different molydate- and tungstate-dependent enzymes in strains carrying mutations in the mod and/or tup genes.
Another tungstate and molybdate uptake system, WtpABC, was first identified in P. furiosus (Bevers et al., 2006). In contrast to the ModABC transporters, which are present in the vast majority of bacteria, the WtpABC transporter appears to be the predominant ABC transporter for these substrates in archaea (Zhang & Gladyshev, 2008). WtpA, the solute receptor, binds tungstate in the low picomolar range, while the dissociation constant for molybdate is about 103-fold higher (Bevers et al., 2006).
Bioinformatics identified a new subclass of putative molybdate transporters (ModA-like) in species of the hyperthermophilic archaeon Pyrobaculum. No experimental data on these systems are currently available (Zhang & Gladyshev, 2008).
A few nitrogen-fixing organisms produce an alternative nitrogenase containing FeVco (a vanadium-containing variant of the aforementioned FeMoco) when molybdenum is unavailable. The natural vanadium source vanadate is not transported by the known molybdate transport systems. A high-affinity vanadate ABC transporter, VupABC, was identified in the genome of the cyanobacterium Anabaena variabilis in the region near the genes encoding V-nitrogenase. It is not found in completely sequenced genomes from other prokaryotes in which a V-nitrogenase is known to operate. Mutational analysis revealed that a vupB mutant cannot produce V-nitrogenase activity at low concentrations of vanadate (Pratte & Thiel, 2006).