The membrane-associated lipoprotein-9 GmpC from Staphylococcus aureus binds the dipeptide GlyMet via side chain interactions

Bacterial dipeptide ABC transporters function to import a wide range of dipeptide substrates. This ability to transport a wide variety of dipeptides is conferred by the cognate substrate binding protein (SBP) of these transporters. SBPs bind dipeptides with little regard for their amino acid content. Here, we report the 1.7 A resolution structure of lipoprotein-9 (SA0422) of Staphylococcus aureus in complex with the dipeptide glycylmethionine. Experimental characterization of the subcellular location of the protein confirmed that SA0422 is an acylated, peripheral membrane protein. This is the first structure determined for an SBP of a Gram-positive dipeptide ABC transporter. Usually, binding of dipeptides occurs in a binding pocket that is largely hydrated and able to accommodate the side chains of several different amino acid residues. Unlike any other known SBP, lipoprotein-9 binds the side chains of the glycylmethionine dipeptide through very specific interactions. Lipoprotein-9 shares significant structural and sequence homology with the MetQ family of methionine SBP. Sequence comparisons between MetQ-like proteins and lipoprotein-9 suggest that the residues forming the tight interactions with the methionine side chains of the ligand are highly conserved between lipoprotein-9 and MetQ homologues, while the residues involved in coordinating the glycine residue are not. Modeling of the Vibrio cholerae MetQ and lipoprotein-9 binding pockets can account for lipoprotein-9 substrate specificity toward glycylmethionine. For this reason, we have designated lipoprotein-9 GmpC, for glycylmethionine binding protein.     

Identification of various substrate-binding proteins of the hyperthermophylic archaeon <Emphasis Type="Italic">Aeropyrum pernix</Emphasis> K1

Proteins from the extracellular medium of Aeropyrum pernix K1 were separated by two-dimensional electrophoresis and identified using mass spectrometry. Six different substrate-binding proteins (SBPs) from the ATP-binding cassette (ABC) transporter family were identified: (1) ABC transporter SBP (Q9YC61); (2) Branched-chain amino-acid ABC transporter, branched-chain amino-acid-binding protein (Q9YDJ6); (3) Oligopeptide ABC transporter, oligopeptide-binding protein (Q9YBL5); (4) Probable ABC transporter SBP (Q9Y9N4); (5) ABC transporter SBP (Q9YBG7); (6) ABC transporter SBP (Q9YFD7). Based on their orthology, division into the following classes was predicted: (1) multiple sugar-transport system SBPs; (2) peptide/nickel-transport system SBPs; and (3) branched-chain amino-acid-transport system SBPs. Further bioinformatic analyses showed that the identified SBPs differ in motif and in transmembrane-domain and signal-peptide organisation. Additionally, for all of these SBPs, sequence homology was found for archaeal proteins, and homologous proteins in bacteria were also found for the ABC transporter SBP Q9YBG7 and the ABC transporter SBP Q9YFD7. This is the first study, where different ABC SBPs from the extracellular medium of A. pernix have been identified using the combined methodology of two-dimensional electrophoresis and mass spectrometry.     

Harnessing natural diversity to probe metabolic pathways

Analyses of cellular processes in the yeast Saccharomyces cerevisiae rely primarily upon a small number of highly domesticated laboratory strains, leaving the extensive natural genetic diversity of the model organism largely unexplored and unexploited. We asked if this diversity could be used to enrich our understanding of basic biological processes. As a test case, we examined a simple trait: the utilization of di/tripeptides as nitrogen sources. The capacity to import small peptides is likely to be under opposing selective pressures (nutrient utilization versus toxin vulnerability) and may therefore be sculpted by diverse pathways and strategies. Hitherto, dipeptide utilization in S. cerevisiae was solely ascribed to the activity of a single protein, the Ptr2p transporter. Using high-throughput phenotyping and several genetically diverse strains, we identified previously unknown cellular activities that contribute to this trait. We find that the Dal5p allantoate/ureidosuccinate permease is also capable of facilitating di/tripeptide transport. Moreover, even in the absence of Dal5p and Ptr2p, an additional activity—almost certainly the periplasmic asparaginase II Asp3p—facilitates the utilization of dipeptides with C-terminal asparagine residues by a different strategy. Another, as-yet-unidentified activity enables the utilization of dipeptides with C-terminal arginine residues. The relative contributions of these activities to the utilization of di/tripeptides vary among the strains analyzed, as does the vulnerability of these strains to a toxic dipeptide. Only by sampling the genetic diversity of multiple strains were we able to uncover several previously unrecognized layers of complexity in this metabolic pathway. High-throughput phenotyping facilitates the rapid exploration of the molecular basis of biological complexity, allowing for future detailed investigation of the selective pressures that drive microbial evolution.     

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.     

Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on β‐mannan possess distinct manno‐oligosaccharide‐binding profiles

Human gut bifidobacteria rely on ATP‐binding cassette (ABC) transporters for oligosaccharide uptake. Multiple oligosaccharide‐specific solute‐binding protein (SBP) genes are occasionally associated with a single ABC transporter, but the significance of this multiplicity remains unclear. Here, we characterize BlMnBP1 and BlMnBP2, the two SBPs associated to the β‐manno‐oligosaccharide (MnOS) ABC transporter in Bifidobacterium animalis subsp. lactis. Despite similar overall specificity and preference to mannotriose (K_d≈80 nM), affinity of BlMnBP1 is up to 2570‐fold higher for disaccharides than BlMnBP2. Structural analysis revealed a substitution of an asparagine that recognizes the mannosyl at position 2 in BlMnBP1, by a glycine in BlMnBP2, which affects substrate affinity. Both substitution types occur in bifidobacterial SBPs, but BlMnBP1‐like variants prevail in human gut isolates. B. animalis subsp. lactis ATCC27673 showed growth on gluco and galactomannans and was able to outcompete a mannan‐degrading Bacteroides ovatus strain in co‐cultures, attesting the efficiency of this ABC uptake system. By contrast, a strain that lacks this transporter failed to grow on mannan. This study highlights SBP diversification as a possible strategy to modulate oligosaccharide uptake preferences of bifidobacterial ABC‐transporters during adaptation to specific ecological niches. Efficient metabolism of galactomannan by distinct bifidobacteria, merits evaluating this plant glycan as a potential prebiotic.     

Domains III and I-2α, at the Entrance of the Binding Cleft,Play an Important Role in Cold Adaptation of the Periplasmic Dipeptide-Binding Protein (DppA) from the Deep-Sea Psychrophilic Bacterium Pseudoalteromonas sp. Strain SM9913

The peptide transporter from a cold-adapted bacterium has never been reported. In the present study, the dpp operon from the psychrophilic bacterium Pseudoalteromonas sp. strain SM9913 was cloned and analyzed. The dipeptide binding protein DppA of SM9913 was overexpressed in Escherichia coli, and its cold adaptation characteristics were studied. The recombinant DppA of SM9913 (PsDppA) displayed the highest ligand-binding affinity at 15°C, whereas the recombinant DppA of E. coli (EcDppA) displayed the highest ligand-binding affinity at 35°C. Thermal and guanidium hydrochloride unfolding analyses indicated that PsDppA has more structural instability than EcDppA. Six domain-exchanged mutants of PsDppA were expressed and purified. Analyses of these mutants indicated that domains III, I-2, and I-3 of PsDppA were less stable than those from EcDppA and that domains III and I-2 made a significant contribution to the high binding affinity of PsDppA at low temperatures. Structural and sequence analyses suggested that the state transition-involved regions in domain III and the α part of domain I-2 are the hot spots of optimization during cold adaptation and that decreasing the side-chain size in these regions is an important strategy for the cold adaptation of PsDppA.The peptide transporter system of bacteria, which plays an important role in nutrition supply, has been extensively investigated, especially for Escherichia coli (9, 11, 20, 21), Salmonella enterica serovar Typhimurium (8, 12, 28, 29), and Lactococcus lactis (2, 13, 14, 25). At present, three peptide transporters—oligopeptide permease (Opp), dipeptide permease (Dpp), and dipeptide and tripeptide permease (Tpp or Dtp)—have been found in bacteria. Dpp proteins are transporters belonging to the ATP-binding cassette (ABC) superfamily and are composed of five subunits: the two integral membrane proteins DppB and DppC forming a permease for substrates, the two cytoplasmic proteins DppD and DppF in charge of ATP hydrolysis, and a periplasmic peptide-binding protein named DppA. At the genetic level, the five genes encoding these five proteins are always organized in an operon named dpp. Dpp has a preference for dipeptides and also transports some tripeptides (25, 27).During peptide translocation, DppA performs identification and binding of substrates and determines the specificity and overall transport parameters for Dpp (27). The crystal structure of DppA from E. coli has been determined, which shows that DppA is composed of three domains (6). The ligand-binding cleft is located between domain I and domain III that are connected by two strands functioning as a hinge. After binding a ligand, the “open” protein turns into a “closed” form, and the ligand is completely buried (6). The tertiary structure of DppA is quite similar to that of OppA (2, 6, 29). It is reported that domain II in OppA is not involved in ligand binding directly, and its role has not been studied in detail (18, 28).Cold-adapted microorganisms are a diverse group living in cold ecosystems, such as the polar and alpine regions and deep sea. Survival in these extreme environments requires the microorganisms to evolve a complex suite of structural and functional adaptations of all cellular constituents, such as membrane, enzymes, energy-generating systems, components responsible for nutrient uptake, and so on (16). There are extensive investigations on modification of membrane lipid composition and adjustment of cold-adapted enzymes (7). However, there has been no report on the peptide transport system from a cold-adapted microorganism. Thus, it is yet unclear how the peptide transport system of a cold-adapted microorganism is adapted to a cold environment.Pseudoalteromonas sp. strain SM9913 is a psychrophilic bacterium isolated from deep-sea sediment, which secretes a large quantity of exopolysaccharide and proteases (5, 23). Our previous studies showed that the exopolysaccharide secreted by strain SM9913 may help the strain enrich the proteinaceous particles and the trace metal ions in the deep-sea environment, and the cold-adapted protease secreted by this strain can degrade various soluble and insoluble proteins to provide nutrients (4, 32). Efficient transport of peptides and amino acids in the deep-sea cold environment is very important for the growth of strain SM9913. In order to elucidate how the peptide transport system of strain SM9913 is adapted to a deep-sea cold environment, the dpp operon of strain SM9913 was cloned and analyzed in the present study. The periplasmic dipeptide-binding protein DppA of Pseudoalteromonas sp. strain SM9913 (PsDppA) was overexpressed in E. coli, and its ligand-binding affinity and structural stability were studied and compared to those of DppA of E. coli (EcDppA). Moreover, by domain-exchanged mutation, the cold adaptation mechanism of PsDppA at the domain level was studied.     

Transcriptional Regulation,Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn²⁺ and Mn²⁺ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn²⁺ or Mn²⁺ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn²⁺ over Mn²⁺ specificity, the Zn²⁺ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn²⁺-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn²⁺-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn²⁺-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn²⁺ specificity.     

Differential regulation and substrate preferences in two peptide transporters of Saccharomyces cerevisiae

Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in Saccharomyces cerevisiae. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [(14)C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides. S. cerevisiae W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [(14)C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of PTR2, DAL5 expression was influenced by the addition of Leu and by the CUP9 gene. However, DAL5 expression was downregulated in the presence of leucine and the absence of CUP9, whereas PTR2 was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.     

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity ( K _m, 2.6 μM) and, although it also binds betaine ( K _m, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX ( K _m, 24 μM) and the betaine-specific SBP BetX ( K _m, 0.6 μM). Unlike most ABC transporter loci, caiX , betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.     

Bacillusd-stereospecific metallo-amidohydrolase: Active-site metal-ion substitution changes substrate specificity

From investigation of 2000 soil isolates, we identified a d-stereospecific metallo-amidohydrolase that can hydrolyze d-aminoacyl derivatives from the culture supernatant of Bacillus sp. 62E11: 62E11DppA. The enzyme binds two equivalents of zinc, exhibits 70% identity with that of d-aminopeptidases from Bacillus subtilis (DppA). In fact, 62E11DppA has strict specificity toward d-aminoacyl derivatives, i.e., the enzyme shows high activity toward d-aminoacyl benzyl esters and little activity toward d-amino acid containing peptides. Moreover, 62E11DppA exhibits a dramatic change in its activity and substrate specificity by substitution of metal ions in its active site. Based on results of kinetic studies using apo-62E11DppA with various metal ion and substrate concentrations, we propose a possible mechanism for the change in its activity and specificity by substitution of metal ions: the substitution of metal ions in 62E11DppA dramatically changes its activity by altering the substrate specificity.     

DtpB (YhiP) and DtpA (TppB, YdgR) are prototypical proton-dependent peptide transporters of Escherichia coli

The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H(+)-dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.     

Dipeptide synthesis by L-amino acid ligase from Ralstonia solanacearum

Despite its utility, dipeptides have not been widely used due to the absence of an efficient manufacturing method. Recently, a novel method for effective production of dipeptides using l-amino acid α-ligase (Lal) is presented. Lal, which is only identified in Bacillus subtilis, catalyzes dipeptide synthesis from unprotected amino acids in an ATP-dependent manner. However, not all the dipeptide can be synthesized by Lal from B. subtilis (BsLal) due to its substrate specificity. Here, we attempted to find a novel Lal exhibiting different substrate specificity from BsLal. By in silico screening based on the amino acid sequence of BsLal, RSp1486a an unknown protein from Ralstonia solanacearum was found to show the Lal activity. RSp1486a exhibited different substrate specificity from BsLal, and preferably synthesized hetero-dipeptides where more bulky amino acid was placed at N terminus and less bulky amino acid was placed at C terminus in opposition to those synthesized by BsLal.     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-expressed the previously uncharacterized YjdL and investigated the peptide specificity by means of uptake inhibition. The IC₅₀ value for the dipeptide Ala-Ala was measured to 22 mM while Ala-Ala-Ala was not able to inhibit uptake. In addition, IC₅₀ values of 0.3 mM and 1.5 mM were observed for Ala-Lys and Tyr-Ala, respectively, while the alanyl-extended tripeptides Ala-Lys-Ala, Ala-Ala-Lys, Ala-Tyr-Ala and Tyr-Ala-Ala displayed values of 8, >50, 31 and 31 mM, respectively. These results clearly indicate that unlike most POT members characterized to date, including YdgR and YhiP, YjdL shows significantly higher specificity towards dipeptides.     

A role for PchHI as the ABC transporter in iron acquisition by the siderophore pyochelin in Pseudomonas aeruginosa

Iron is an essential nutrient for bacterial growth but poorly bioavailable. Bacteria scavenge ferric iron by synthesizing and secreting siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. After capturing a ferric iron molecule, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and then by the inner membrane permease FptX. Here, using molecular biology, ⁵⁵Fe uptake assays, and LC–MS/MS quantification, we first find a role for PchHI as the heterodimeric ABC transporter involved in the siderophore-free iron uptake into the bacterial cytoplasm. We also provide the first evidence that PCH is able to reach the bacterial periplasm and cytoplasm when both FptA and FptX are expressed. Finally, we detected an interaction between PchH and FptX, linking the ABC transporter PchHI with the inner permease FptX in the PCH-Fe uptake pathway. These results pave the way for a better understanding of the PCH siderophore pathway, giving future directions to tackle P. aeruginosa infections.     

AtPTR1 and AtPTR5 transport dipeptides in planta

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.     

Solute-binding protein-dependent ABC transporters are responsible for solute efflux in addition to solute uptake

The ATP-binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to manQ1. The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible. However, contrary to this paradigm, we show that three solute-binding protein-dependent (SBP) ABC transporters of amino acids, i.e. the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium, are bidirectional, being responsible for efflux in addition to the uptake of solutes. The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated. SBP ABC transporters promote active uptake because, although the Vmax values for uptake and efflux are not significantly different, there is a 103-104 higher affinity for uptake of solute compared with efflux. Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells.     

Homes for the orphans: utilization of multiple substrate-binding proteins by ABC transporters

Acquiring nutrients from the environment is essential for all microbes, and the ATP-binding cassette (ABC) transporters are one of the major routes by which bacteria achieve it. In this issue of Molecular Microbiology , Chen et al. describe their characterization of what appeared at first glance a simple ABC transporter for acquisition of quaternary ammonium compounds (QACs) in Pseudomonas sp., but their persistence in fully determining the properties of this system led to the experimental demonstration that QAC uptake utilizes three different substrate-binding proteins (SBPs), two of which are encoded at remote locations on the genome as 'orphan' SBPs that are each able to function with a single core ABC transporter. Building on the unusual nature of this system, in which multiple SBPs with non-overlapping substrate specificities compete for the same transporter binding site, they designed elegant in vivo experiments that suggest that only substrate-bound SBPs are able to form functional complexes with the membrane domains. This new finding provides an important piece of in vivo data leading to further insight into how this ubiquitous family of transporters operates.