Several Archaeal Homologs of Putative Oligopeptide-Binding Proteins Encoded by Thermotoga maritima Bind Sugars

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the β-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), β-1,4-mannobiose; and TM1226 (ManD), β-1,4-mannobiose, β-1,4-mannotriose, β-1,4-mannotetraose, β-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), α-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.     

Oligosaccharide binding proteins from Bifidobacterium longum subsp. infantis reveal a preference for host glycans

Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process.     

Substrate adaptabilities of Thermotogae mannan binding proteins as a function of their evolutionary histories

The Thermotogae possess a large number of ATP-binding cassette (ABC) transporters, including two mannan binding proteins, ManD and CelE (previously called ManE). We show that a gene encoding an ancestor of these was acquired by the Thermotogae from the archaea followed by gene duplication. To address the functional evolution of these proteins as a consequence of their evolutionary histories, we measured the binding affinities of ManD and CelE orthologs from representative Thermotogae. Both proteins bind cellobiose, cellotriose, cellotetraose, β-1,4-mannotriose, and β-1,4-mannotetraose. The CelE orthologs additionally bind β-1,4-mannobiose, laminaribiose, laminaritriose and sophorose while the ManD orthologs additionally only weakly bind β-1,4-mannobiose. The CelE orthologs have higher unfolding temperatures than the ManD orthologs. An examination of codon sites under positive selection revealed that many of these encode residues located near or in the binding site, suggesting that the proteins experienced selective pressures in regions that might have changed their functions. The gene arrangement, phylogeny, binding properties, and putative regulatory networks suggest that the ancestral mannan binding protein was a CelE ortholog which gave rise to the ManD orthologs. This study provides a window on how one class of proteins adapted to new functions and temperatures to fit the physiologies of their new hosts.     

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.     

The crystal structure of the family 6 carbohydrate binding module from Cellvibrio mixtus endoglucanase 5a in complex with oligosaccharides reveals two distinct binding sites with different ligand specificities

Glycoside hydrolases that release fixed carbon from the plant cell wall are of considerable biological and industrial importance. These hydrolases contain non-catalytic carbohydrate binding modules (CBMs) that, by bringing the appended catalytic domain into intimate association with its insoluble substrate, greatly potentiate catalysis. Family 6 CBMs (CBM6) are highly unusual because they contain two distinct clefts (cleft A and cleft B) that potentially can function as binding sites. Henshaw et al. (Henshaw, J., Bolam, D. N., Pires, V. M. R., Czjzek, M., Henrissat, B., Ferreira, L. M. A., Fontes, C. M. G. A., and Gilbert, H. J. (2003) J. Biol. Chem. 279, 21552-21559) show that CmCBM6 contains two binding sites that display both similarities and differences in their ligand specificity. Here we report the crystal structure of CmCBM6 in complex with a variety of ligands that reveals the structural basis for the ligand specificity displayed by this protein. In cleft A the two faces of the terminal sugars of beta-linked oligosaccharides stack against Trp-92 and Tyr-33, whereas the rest of the binding cleft is blocked by Glu-20 and Thr-23, residues that are not present in CBM6 proteins that bind to the internal regions of polysaccharides in cleft A. Cleft B is solvent-exposed and, therefore, able to bind ligands because the loop, which occludes this region in other CBM6 proteins, is much shorter and flexible (lacks a conserved proline) in CmCBM6. Subsites 2 and 3 of cleft B accommodate cellobiose (Glc-beta-1,4-Glc), subsite 4 will bind only to a beta-1,3-linked glucose, whereas subsite 1 can interact with either a beta-1,3- or beta-1,4-linked glucose. These different specificities of the subsites explain how cleft B can accommodate beta-1,4-beta-1,3- or beta-1,3-beta-1,4-linked gluco-configured ligands.     

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.     

Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein-ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure-function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino‐acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequence-based methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria.     

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix

The archaeon Aeropyrum pernix grows optimally at 90°C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA_Ap) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA_Ap-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA_Ap was about 70% at 90°C using a 1/100 (w/w) OppA_Ap/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA_Ap with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA_Ap as the extracellular component for the uptake of oligopeptides.     

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.     

A Highly Selective Oligopeptide Binding Protein from the Archaeon Sulfolobus Solfataricus

SSO1273 of Sulfolobus solfataricus was identified as a cell surface-bound protein by a proteomics approach. Sequence inspection of the genome revealed that the open reading frame of sso1273 is associated in an operon-like structure with genes encoding all the remaining components of a canonical protein-dependent ATP-binding cassette (ABC) transporter. sso1273 gene expression and SSO1273 protein accumulation on the cell surface were demonstrated to be strongly induced by the addition of a peptide mixture (tryptone) to the culture medium. The native protein was obtained in multimeric form, mostly hexameric, under the purification conditions used, and it was characterized as an oligopeptide binding protein, named S. solfataricus OppA (OppA_Ss). OppaA_Ss possesses typical sequence patterns required for glycosylphosphatidylinositol lipid anchoring, resulting in an N-linked glycoprotein with carbohydrate moieties likely composed of high mannose and/or hybrid complex carbohydrates. OppA_Ss specifically binds oligopeptides and shows a marked selectivity for the amino acid composition of substrates when assayed in complex peptide mixtures. Moreover, a truncated version of OppA_Ss, produced in recombinant form and including the putative binding domain, showed a low but significant oligopeptide binding activity.Sulfolobus solfataricus is an obligate aerobe that grows in hot and acidic environments either chemolithotrophically by oxidizing metal cations (Fe²⁺ or S⁻) or heterotrophically on simple sugars. It originates from a solfataric field with temperatures between 75°C and 90°C and pH values of 1.0 to 3.0 (9, 15). Within its environment, Sulfolobus can interact with a complex ecosystem consisting of a variety of primary producers and decomposers of organic matter. Moreover, biotopes such as the solfataric field of Sulfolobus contain decomposing materials of higher plants, including cellulose, starch, and proteinaceous compounds, that can act as potential carbon sources. Although S. solfataricus has been reported to grow on a wide variety of reduced organic compounds as the sole carbon and energy source (15), the nutrient utilization by this microorganism requires complex mechanisms of uptake and metabolism that are not yet well defined.Numerous efforts have been directed toward the identification of the carbohydrate utilization strategy in this hyperthermophilic archaeon (18, 23). The metabolic pathways for the degradation of a variety of sugars have been studied in detail and provide evidence that S. solfataricus predominantly uses binding-protein-dependent ABC transporters for the uptake of carbohydrate compounds (1, 2, 13).Archaeal ABC uptake systems are divided into two main classes: the carbohydrate (CUT) and the di-/oligopeptide uptake transporter classes (2). These transporter families use ATP hydrolysis to drive a unidirectional accumulation of solutes into the cytoplasm. The translocator components are composed of two integral membrane proteins, two peripheral membrane proteins that bind and hydrolyze ATP, and an extracellular substrate-binding protein (SBP). The SBP subunit captures and delivers the substrate to the translocon, and it is therefore considered to be one of the determinants of the transport specificity (2, 7, 10).All sequenced genomes of archaea and thermophilic bacteria contain a large number of genes encoding putative ABC transport systems involved in the uptake of organic solutes. The preference of hyperthermophiles for ABC-type transporters could be important for the survival strategy in their natural habitat. In the nutrient-poor environments, such as hydrothermal vents or sulfuric hot springs, in which these organisms thrive, ABC transporters have the advantage that they can scavenge solutes at very low concentrations due to the high binding affinities of their SBP components. Furthermore, these transporters can catalyze translocation at a high rate, resulting in high internal concentrations of solutes. In contrast, secondary transport systems exhibit lower binding affinities, which make these systems less suitable for growth in extreme environments.So far, attempts to predict the functional specificity of the ABC transporters using computational tools have been largely unsuccessful (2, 13, 20). For example, some characterized archaeal sugar transporters, based on the sequence identity and domain organization, were predicted to be di-/oligopeptide transporters (13, 20). These include the cellobiose/β-glucoside transporter system of Pyrococcus furiosus (20) and the maltose/maltodextrin and cellobiose/cello-oligomer transporters of S. solfataricus (13). However, genes encoding sugar-metabolizing enzymes are located in the vicinity of all these transport systems, suggesting that the location of the ABC operon can support the specific transport function.Like oligopeptide binding proteins, MalE and CbtA bind a broad range of polymeric substrates (13, 20). In contrast, sugar-binding proteins usually exhibit a narrow substrate specificity that is often limited to monosaccharides. Therefore, it may well be that the substrate binding pocket of CbtA and MalE resembles that of the OppA family of binding proteins that can accommodate a range of short and long oligopeptides.S. solfataricus contains 37 putative ABC transporters at the genome level (TransportDB, Genomic Comparisons of Membrane Transport systems [http://www.membranetransport.org/index.html]), but only a few of these systems have been functionally characterized. It is interesting that all of these are implicated in the uptake of mono-/oligosaccharides (1, 13, 20, 25).The present work describes the isolation and characterization of the first functional ABC substrate binding protein from S. solfataricus belonging to the di-/oligopeptide transporter family, named S. solfataricus OppA (OppA_Ss). We demonstrate that OppA_Ss is an outer-cell-surface-anchored protein and that its expression is highly induced in the presence of a source of peptides in the culture broth. Furthermore, in vitro substrate specificity studies using complex oligopeptide mixtures indicate that OppA_Ss is highly selective in peptide recognition.     

Carbohydrate transporting membrane proteins of the rumen bacterium, Butyrivibrio proteoclasticus

The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation.     

Bioenergetics and solute uptake under extreme conditions

The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms. The proton and sodium permeabilities of membranes increase with temperature. Psychrophilic and mesophilic bacteria and mesophilic, (hyper)thermophilic, and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains constant (homeoproton permeability). Thermophilic bacteria are an exception. They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes. Transport of solutes across bacterial and archaeal membranes is mainly catalyzed by primary ATP-driven transport systems or by proton- or sodium-motive-force-driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary uptake systems. Several high-affinity ATP-binding cassette (ABC) transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.     

ABC transporters: one,two or four extracytoplasmic substrate-binding sites?

Two families of ATP-binding cassette (ABC) transporters in which one or two extracytoplasmic substrate-binding domains are fused to either the N- or C-terminus of the translocator protein have been detected. This suggests that two, or even four, substrate-binding sites may function in the ABC transporter complex. This domain organization in ABC transporters, widely represented among microorganisms, raises new possibilities for how the substrate-binding protein(s) (SBPs) might interact with the translocator. One appealing hypothesis is that multiple substrate-binding sites in proximity to the entry site of the translocation pore enhance the transport capacity. We also discuss the implications of multiple substrate-binding sites in close proximity to the translocator in terms of broadened substrate specificity and possible cooperative interactions between SBPs and the translocator.     

Transport capabilities of eleven gram-positive bacteria: Comparative genomic analyses

The genomes of eleven Gram-positive bacteria that are important for human health and the food industry, nine low G + C lactic acid bacteria and two high G + C Gram-positive organisms, were analyzed for their complement of genes encoding transport proteins. Thirteen to 18% of their genes encode transport proteins, larger percentages than observed for most other bacteria. All of these bacteria possess channel proteins, some of which probably function to relieve osmotic stress. Amino acid uptake systems predominate over sugar and peptide cation symporters, and of the sugar uptake porters, those specific for oligosaccharides and glycosides often outnumber those for free sugars. About 10% of the total transport proteins are constituents of putative multidrug efflux pumps with Major Facilitator Superfamily (MFS)-type pumps (55%) being more prevalent than ATP-binding cassette (ABC)-type pumps (33%), which, however, usually greatly outnumber all other types. An exception to this generalization is Streptococcus thermophilus with 54% of its drug efflux pumps belonging to the ABC superfamily and 23% belonging each to the Multidrug/Oligosaccharide/Polysaccharide (MOP) superfamily and the MFS. These bacteria also display peptide efflux pumps that may function in intercellular signalling, and macromolecular efflux pumps, many of predictable specificities. Most of the bacteria analyzed have no pmf-coupled or transmembrane flow electron carriers. The one exception is Brevibacterium linens, which in addition to these carriers, also has transporters of several families not represented in the other ten bacteria examined. Comparisons with the genomes of organisms from other bacterial kingdoms revealed that lactic acid bacteria possess distinctive proportions of recognized transporter types (e.g., more porters specific for glycosides than reducing sugars). Some homologues of transporters identified had previously been identified only in Gram-negative bacteria or in eukaryotes. Our studies reveal unique characteristics of the lactic acid bacteria such as the universal presence of genes encoding mechanosensitive channels, competence systems and large numbers of sugar transporters of the phosphotransferase system. The analyses lead to important physiological predictions regarding the preferred signalling and metabolic activities of these industrially important bacteria.     

Transport capabilities of eleven gram-positive bacteria: comparative genomic analyses

The genomes of eleven Gram-positive bacteria that are important for human health and the food industry, nine low G+C lactic acid bacteria and two high G+C Gram-positive organisms, were analyzed for their complement of genes encoding transport proteins. Thirteen to 18% of their genes encode transport proteins, larger percentages than observed for most other bacteria. All of these bacteria possess channel proteins, some of which probably function to relieve osmotic stress. Amino acid uptake systems predominate over sugar and peptide cation symporters, and of the sugar uptake porters, those specific for oligosaccharides and glycosides often outnumber those for free sugars. About 10% of the total transport proteins are constituents of putative multidrug efflux pumps with Major Facilitator Superfamily (MFS)-type pumps (55%) being more prevalent than ATP-binding cassette (ABC)-type pumps (33%), which, however, usually greatly outnumber all other types. An exception to this generalization is Streptococcus thermophilus with 54% of its drug efflux pumps belonging to the ABC superfamily and 23% belonging each to the Multidrug/Oligosaccharide/Polysaccharide (MOP) superfamily and the MFS. These bacteria also display peptide efflux pumps that may function in intercellular signalling, and macromolecular efflux pumps, many of predictable specificities. Most of the bacteria analyzed have no pmf-coupled or transmembrane flow electron carriers. The one exception is Brevibacterium linens, which in addition to these carriers, also has transporters of several families not represented in the other ten bacteria examined. Comparisons with the genomes of organisms from other bacterial kingdoms revealed that lactic acid bacteria possess distinctive proportions of recognized transporter types (e.g., more porters specific for glycosides than reducing sugars). Some homologues of transporters identified had previously been identified only in Gram-negative bacteria or in eukaryotes. Our studies reveal unique characteristics of the lactic acid bacteria such as the universal presence of genes encoding mechanosensitive channels, competence systems and large numbers of sugar transporters of the phosphotransferase system. The analyses lead to important physiological predictions regarding the preferred signalling and metabolic activities of these industrially important bacteria.