Substrate-binding Protein Research Articles

Bioinformatics analysis and biochemical characterisation of ABC transporter-associated periplasmic substrate-binding proteins ModA and MetQ from Helicobacter pylori strain SS1

The human gastric pathogen Helicobacter pylori relies on the uptake of host-provided nutrients for its proliferation and pathogenicity. ABC transporters that mediate import of small molecules into the cytoplasm of H. pylori employ their cognate periplasmic substrate-binding proteins (SBPs) for ligand capture in the periplasm. The genome of the mouse-adapted strain SS1 of H. pylori encodes eight ABC transporter-associated SBPs, but little is known about their specificity or structure. In this study, we demonstrated that the SBP annotated as ModA binds molybdate (MoO42−, KD = 3.8 nM) and tungstate (WO42−, KD = 7.8 nM). In addition, we showed that MetQ binds D-methionine (KD = 9.5 μM), but not L-methionine, which suggests the existence of as yet unknown pathway for L-methionine uptake. Homology modelling has led to identification of the ligand-binding residues.

Structural Flexibility of Peripheral Loops and Extended C-terminal Domain of Short Length Substrate Binding Protein from Rhodothermus marinus.

Substrate binding proteins (SBPs) bind to specific ligands in the periplasmic regions of cells and then bind to membrane proteins to participate in transport or signal transduction. Typically, SBPs consist of two α/β domains and recognize the substrate by a flexible hinge region between the two domains. Conversely, the short-length SBPs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes. We previously determined the crystal structure of Rhodothermus marinus SBP (named as RmSBP), consisting of a single α/β domain; however, the substrate recognition mechanism is still unclear. To better understand the functions of short length RmSBP, we performed a comprehensive study, involving comparative structure analysis, computational substrate docking, and X-ray crystallographic data. RmSBP shares a high level of similarity in the α/β domain region with other SBPs, but it has a distinct topology in the C-terminal domain. The substrate binding model suggested that conformational changes in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility in the β1–α2 loop, β5–β6 loop, and extended C-terminal domains, based on the electron density map and temperature B-factor analysis. These results provide information that will further our understanding on the functions of the short length SBP.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10930-021-09970-z.

Structural basis for a bacterial Pip system plant effector recognition protein

A number of plant-associated proteobacteria have LuxR family transcription factors that we refer to as PipR subfamily members. PipR proteins play roles in interactions between bacteria and their plant hosts, and some are important for bacterial virulence of plants. We identified an ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), as a potent effector of PipR-mediated gene regulation in the plant endophyte Pseudomonas GM79. HEHEAA-dependent PipR activity requires an ATP-binding cassette-type active transport system, and the periplasmic substrate-binding protein (SBP) of that system binds HEHEAA. To begin to understand the molecular basis of PipR system responses to plant factors we crystallized a HEHEAA-responsive SBP in the free- and HEHEAA-bound forms. The SBP, which is similar to peptide-binding SBPs, was in a closed conformation. A narrow cavity at the interface of its two lobes is wide enough to bind HEHEAA, but it cannot accommodate peptides with side chains. The polar atoms of HEHEAA are recognized by hydrogen-bonding interactions, and additional SBP residues contribute to the binding site. This binding mode was confirmed by a structure-based mutational analysis. We also show that a closely related SBP from the plant pathogen Pseudomonas syringae pv tomato DC3000 does not recognize HEHEAA. However, a single amino acid substitution in the presumed effector-binding pocket of the P. syringae SBP converted it to a weak HEHEAA-binding protein. The P. syringae PipR depends on a plant effector for activity, and our findings imply that different PipR-associated SBPs bind different effectors.

The Role of a Dipeptide Transporter in the Virulence of Human Pathogen, Helicobacter pylori.

Helicobacter pylori harbors a dipeptide (Dpp) transporter consisting of a substrate-binding protein (DppA), two permeases (DppB and C), and two ATPases (DppD and F). The Dpp transporter is responsible for the transportation of dipeptides and short peptides. We found that its expression is important for the growth of H. pylori. To understand the role of the Dpp transporter in the pathogenesis of H. pylori, the expression of virulence factors and H. pylori-induced IL-8 production were investigated in H. pylori wild-type and isogenic H. pylori Dpp transporter mutants. We found that expression of CagA was downregulated, while expression of type 4 secretion system (T4SS) components was upregulated in Dpp transporter mutants. The DppA mutant strain expressed higher levels of outer membrane proteins (OMPs), including BabA, HopZ, OipA, and SabA, and showed a higher adhesion level to gastric epithelial AGS cells compared with the H. pylori 26695 wild-type strain. After infection of AGS cells, H. pylori ΔdppA induced a higher level of NF-κB activation and IL-8 production compared with wild-type. These results suggested that in addition to supporting the growth of H. pylori, the Dpp transporter causes bacteria to alter the expression of virulence factors and reduces H. pylori-induced NF-κB activation and IL-8 production in gastric epithelial cells.

A Systematic Review of Campylobacter jejuni Vaccine Candidates for Chickens.

Campylobacter jejuni infection linked to the consumption of contaminated poultry products is one of the leading causes of human enteric illness worldwide. Vaccination of chickens is one of the potential strategies that could be used to control C. jejuni colonization. To date, various C. jejuni vaccines using potential antigens have been evaluated, but a challenge in identifying the most effective formulation is the wide variability in vaccine efficacies reported. A systematic review was undertaken to compare C. jejuni vaccine studies. Based upon specific selection criteria eligible papers were identified and included in the analysis. Vaccine efficacy reported from different C. jejuni antigens, vaccine types, and vaccination regimens reported in these papers were reviewed. Our analysis shows that total outer membrane proteins and cysteine ABC transporter substrate-binding protein were among the most efficacious vaccine antigen candidates reported. This review also highlights the importance of the need for increased consistency in the way C. jejuni vaccine studies in poultry are designed and reported in order to be able to undertake a robust comparison of C. jejuni vaccine candidates.

The Role of Calcium in Regulating the Conformational Dynamics of d-Galactose/d-Glucose-Binding Protein Revealed by Markov State Model Analysis.

The d-glucose/d-galactose-binding protein (GGBP) from Escherichia coli is a substrate-binding protein (SBP) associated with sugar transport and chemotaxis. It is also a calcium-binding protein, which makes it unique in the SBP family. However, the functional importance of Ca2+ binding is not fully understood. Here, the calcium-dependent properties of GGBP were explored by all-atom molecular dynamics simulations and Markov state model (MSM) analysis as well as single-molecule Förster resonance energy transfer (smFRET) measurements. In agreement with previous experimental studies, we observed the structure stabilization effect of Ca2+ binding on the C-terminal domain of GGBP, especially the Ca2+-binding site. Interestingly, the MSMs of calcium-depleted GGBP and calcium-bound GGBP (GGBP/Ca2+) demonstrate that Ca2+ greatly stabilizes the open conformation, and smFRET measurements confirmed this result. Further analysis reveals that Ca2+ binding disturbs the local hydrogen bonding interactions and the conformational dynamics of the hinge region, thereby weakening the long-range interdomain correlations to favor the open conformation. These results suggest an active regulatory role of Ca2+ binding in GGBP, which finely tunes the conformational distribution. The work sheds new light on the study of calcium-binding proteins in prokaryotes.

Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activity.

Biosynthetic gene clusters (BGCs) are operonic sets of microbial genes that synthesize specialized metabolites with diverse functions, including siderophores and antibiotics, which often require export to the extracellular environment. For this reason, genes for transport across cellular membranes are essential for the production of specialized metabolites and are often genomically colocalized with BGCs. Here, we conducted a comprehensive computational analysis of transporters associated with characterized BGCs. In addition to known exporters, in BGCs we found many importer-specific transmembrane domains that co-occur with substrate binding proteins possibly for uptake of siderophores or metabolic precursors. Machine learning models using transporter gene frequencies were predictive of known siderophore activity, molecular weights, and a measure of lipophilicity (log P) for corresponding BGC-synthesized metabolites. Transporter genes associated with BGCs were often equally or more predictive of metabolite features than biosynthetic genes. Given the importance of siderophores as pathogenicity factors, we used transporters specific for siderophore BGCs to identify both known and uncharacterized siderophore-like BGCs in genomes from metagenomes from the infant and adult gut microbiome. We find that 23% of microbial genomes from premature infant guts have siderophore-like BGCs, but only 3% of those assembled from adult gut microbiomes do. Although siderophore-like BGCs from the infant gut are predominantly associated with Enterobacteriaceae and Staphylococcus, siderophore-like BGCs can be identified from taxa in the adult gut microbiome that have rarely been recognized for siderophore production. Taken together, these results show that consideration of BGC-associated transporter genes can inform predictions of specialized metabolite structure and function.

Triggering Closure of a Sialic Acid TRAP Transporter Substrate Binding Protein through Binding of Natural or Artificial Substrates

The pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.

Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway.

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.

Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability

ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.

Identification of differentially expressed Legionella genes during its intracellular growth in Acanthamoeba

Legionella grows intracellularly in free-living amoeba as well as in mammalian macrophages. Until now, the overall gene expression pattern of intracellular Legionella in Acanthamoeba was not fully explained. Intracellular bacteria are capable of not only altering the gene expression of its host, but it can also regulate the expression of its own genes for survival. In this study, differentially expressed Legionella genes within Acanthamoeba during the 24 h intracellular growth period were investigated for comparative analysis. RNA sequencing analysis revealed 3,003 genes from the intracellular Legionella. Among them, 115 genes were upregulated and 1,676 genes were downregulated more than 2 fold compared to the free Legionella. Gene ontology (GO) analysis revealed the suppression of multiple genes within the intracellular Legionella, which were categorized under ‘ATP binding’ and ‘DNA binding’ in the molecular function domain. Gene expression of alkylhydroperoxidase, an enzyme involved in virulence and anti-oxidative stress response, was strongly enhanced 24 h post-intracellular growth. Amino acid ABC transporter substrate-binding protein that utilizes energy generation was also highly expressed. Genes associated with alkylhydroperoxidase, glucose pathway, and Dot/Icm type IV secretion system were shown to be differentially expressed. These results contribute to a better understanding of the survival strategies of intracellular Legionella within Acanthamoeba.

Identification of novel vaccine candidates against carbapenem resistant Klebsiella pneumoniae: A systematic reverse proteomic approach

Klebsiella pneumoniae is declared as antibiotic resistant by WHO, with the critical urgency of developing novel antimicrobial therapeutics as drug resistance is the second most dangerous threat after terrorism. Besides many attempts still, there is no effective vaccine available against K. pneumoniae. By utilizing all the available proteomic data we prioritized the novel proteins ideal for vaccine development using bioinformatics tools and techniques. Among the huge data, eight proteins passed all the barriers and were considered ideal candidates for vaccine development. These include: copper silver efflux system outer membrane protein (CusC), outer membrane porin protein (OmpN), Fe++ enterobactin transporter substrate binding protein (fepB), zinc transporter substrate binding protein (ZnuA), ribonuclease HI, tellurite resistant methyltransferase (the B), and two uncharacterized hypothetical proteins (WP_002918223 and WP_002892366). These proteins were also subjected to epitope analysis and were found best for developing subunit vaccine against K. pneumoniae. The study shows that the potential vaccine targets are sufficiently efficient being virulent, of outer membranous origin and can be proposed for the DNA third-generation vaccines development that would help to cope up infections caused by multidrug-resistant K. pneumoniae.

The Salmonella enterica serovar Typhimurium virulence factor STM3169 is a hexuronic acid binding protein component of a TRAP transporter.

The intracellular pathogen S. Typhimurium is a leading cause of foodborne illness across the world and is known to rely on a range of virulence factors to colonize the human host and cause disease. The gene coding for one such factor, stm3169, was determined to be upregulated upon macrophage entry and its disruption reduces survival in the macrophage. In this study we characterize the STM3169 protein, which forms the substrate binding protein (SBP) of an uncharacterized tripartite ATP-independent periplasmic (TRAP) transporter. Genome context analysis of the genes encoding this system in related bacteria suggests a function in sugar acid transport. We demonstrate that purified STM3169 binds d-glucuronic acid with high affinity and specificity. S. Typhimurium LT2 can use this sugar acid as a sole carbon source and the genes for a probable catabolic pathway are present in the genome. As this gene was previously implicated in macrophage survival, it suggests a role for d-glucuronate as an important carbon source for S. Typhimurium in this environment.

Conformational Trapping of a β-Glucosides-Binding Protein Unveils the Selective Two-Step Ligand-Binding Mechanism of ABC Importers

Substrate-binding proteins (SBPs), selectively capture ligand(s) and ensure their translocation via its cognate ATP-binding cassette (ABC) import system. SBPs bind their cognate ligand(s) via an induced-fit mechanism known as the “Venus Fly-trap”; however, this mechanism lacks the atomic details of all conformational landscape as the confirmatory evidence(s) in its support. In this study, we delineate the atomic details of an SBP, β-glucosides-binding protein (βGlyBP) from Thermus thermophilus HB8. The protein βGlyBP is multi-specific and binds to different types of β-glucosides varying in their glycosidic linkages viz. β-1,2; β-1,3; β-1,4 and β-1,6 with a degree of polymerization of 2–5 glucosyl units. Structurally, the protein βGlyBP possesses four subdomains (N1, N2, C1 and C2). The unliganded protein βGlyBP remains in an open state, which closes upon binding to sophorose (SOP2), laminari-oligosaccharides (LAMn), cello-oligosaccharides (CELn), and gentiobiose (GEN2). This study reports, for the first time, four different structural states (open-unliganded, partial-open-unliganded, open-liganded and closed-liganded) of the protein βGlyBP, revealing its conformational changes upon ligand binding and suggesting a two-step induced-fit mechanism. Further, results suggest that the conformational changes of N1 and C1 subdomains drive the ligand binding, unlike that of the whole N- and C-terminal domains (NTD and CTD) as known in the “Venus Fly-trap” mechanism. Additionally, profiling of stereo-selection mechanism for α- and β-glucosides reveals that in the ligand-binding site four secondary structural elements (L1, H1, H2 and H3) drive the ligand selection. In summary, results demonstrate that the details of conformational changes and ligand selection are pre-encoded in the SBPs.

Kinetic Modelling of Transport Inhibition by Substrates in ABC Importers

Prokaryotic ATP-binding cassette (ABC) importers require a substrate-binding protein (SBP) for the capture and delivery of the cognate substrate to the transmembrane domain (TMD) of the transporter. Various biochemical compounds have been identified that bind to the SBP but are not transported. The mechanistic basis for the “non-cognate” substrates not being transported differs. Some non-cognate substrates fail to trigger the appropriate conformational change in the SBP, resulting in loss of affinity for the TMD or the inability to allosterically activate transport. In another mechanism, the SBP cannot release the bound non-cognate substrate. Here, we used rate equations to derive the steady-state transport rate of cognate substrates of an ABC importer and investigated how non-cognate substrates influence this rate. We found that under limiting non-cognate substrate concentrations, the transport rate remains unaltered for each of the mechanisms. In contrast, at saturating substrate and SBP concentrations, the effect of the non-cognate substrate depends heavily on the respective mechanism. For instance, the transport rate becomes zero when the non-cognate substrate cannot be released by the SBP. Yet it remains unaffected when substrate release is possible but the SBP cannot dock onto the TMDs. Our work shows how the different mechanisms of substrate inhibition impact the transport kinetics, which is relevant for understanding and manipulating solute fluxes and hence the propagation of cells in nutritionally complex milieus.

Ancient co-option of an amino acid ABC transporter locus in Pseudomonas syringae for host signal-dependent virulence gene regulation.

Pathogenic bacteria frequently acquire virulence traits via horizontal gene transfer, yet additional evolutionary innovations may be necessary to integrate newly acquired genes into existing regulatory pathways. The plant bacterial pathogen Pseudomonas syringae relies on a horizontally acquired type III secretion system (T3SS) to cause disease. T3SS-encoding genes are induced by plant-derived metabolites, yet how this regulation occurs, and how it evolved, is poorly understood. Here we report that the two-component system AauS-AauR and substrate-binding protein AatJ, proteins encoded by an acidic amino acid-transport (aat) and -utilization (aau) locus in P. syringae, directly regulate T3SS-encoding genes in response to host aspartate and glutamate signals. Mutants of P. syringae strain DC3000 lacking aauS, aauR or aatJ expressed lower levels of T3SS genes in response to aspartate and glutamate, and had decreased T3SS deployment and virulence during infection of Arabidopsis. We identified an AauR-binding motif (Rbm) upstream of genes encoding T3SS regulators HrpR and HrpS, and demonstrated that this Rbm is required for maximal T3SS deployment and virulence of DC3000. The Rbm upstream of hrpRS is conserved in all P. syringae strains with a canonical T3SS, suggesting AauR regulation of hrpRS is ancient. Consistent with a model of conserved function, an aauR deletion mutant of P. syringae strain B728a, a bean pathogen, had decreased T3SS expression and growth in host plants. Together, our data suggest that, upon acquisition of T3SS-encoding genes, a strain ancestral to P. syringae co-opted an existing AatJ-AauS-AauR pathway to regulate T3SS deployment in response to specific host metabolite signals.

Characteristics of three microbial colonization states in the duodenum of the cirrhotic patients.

Aim: Investigation of characteristics of different duodenal microbial colonization states in patients with liver cirrhosis (LC). Materials & methods: Deep-sequencing analyses of the 16S rRNA gene V1-V3 regions were performed. Results: Both bacterial compositions and richness were different between the three-clustered LC microbiotas, in other words, Cluster_1_LC, Cluster_2_LC and Cluster_3_LC. Cluster_1_LC were more likely at severe dysbiosis status due to its lowest modified cirrhosis dysbiosis ratio. OTU12_Prevotella and OTU10_Comamonas were most associated with Cluster_1_LC and Cluster_3_LC, respectively, while OTU38_Alloprevotella was vital in Cluster_2_LC. Pyruvate-ferredoxin/flavodoxin oxidoreductase, dihydroorotate dehydrogenase and branched-chain amino acid transport system substrate-binding protein were most associated with Cluster_1_LC, Cluster_2_LC and Cluster_3_LC, respectively. Conclusion: The three duodenal microbial colonization states had distinct representative characteristics, which might reflect the health status of cirrhotic patients.

Multiple Genes of Symbiotic Plasmid and Chromosome in Type II Peanut Bradyrhizobium Strains Corresponding to the Incompatible Symbiosis With Vigna radiata.

Rhizobia are capable of establishing compatible symbiosis with their hosts of origin and plants in the cross-nodulation group that the hosts of origin belonged to. However, different from the normal peanut Bradyrhizobium (Type I strains), the Type II strains showed incompatible symbiosis with Vigna radiata. Here, we employed transposon mutagenesis to identify the genetic loci related to this incompatibility in Type II strain CCBAU 53363. As results, seven Tn5 transposon insertion mutants resulted in an increase in nodule number on V. radiata. By sequencing analysis of the sequence flanking Tn5 insertion, six mutants were located in the chromosome of CCBAU 53363, respectively encoding acyltransferase (L265) and hypothetical protein (L615)—unique to CCBAU 53363, two hypothetical proteins (L4 and L82), tripartite tricarboxylate transporter substrate binding protein (L373), and sulfur oxidation c-type cytochrome SoxA (L646), while one mutant was in symbiotic plasmid encoding alanine dehydrogenase (L147). Significant differences were observed in L147 gene sequences and the deduced protein 3D structures between the Type II (in symbiotic plasmid) and Type I strains (in chromosome). Conversely, strains in both types shared high homologies in the chromosome genes L373 and L646 and in their protein 3D structures. These data indicated that the symbiotic plasmid gene in Type II strains might have directly affected their symbiosis incompatibility, whereas the chromosome genes might be indirectly involved in this process by regulating the plasmid symbiosis genes. The seven genes may initially explain the complication associated with symbiotic incompatibility.

Degradative Capacity of Two Strains of Rhodonia placenta: From Phenotype to Genotype

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context.

1H, 13C, 15N backbone resonance assignments of the apo and holo forms of the ABC transporter solute binding protein PiuA from Streptococcus pneumoniae

Streptococcus pneumoniae is a Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Iron acquisition is essential for its survival and virulence, especially under host-imposed nutritional immunity. S. pneumoniae expresses several ATP-binding cassette (ABC) transporters to facilitate acquisition under iron limitation, including PitABCD, PiaABCD, and PiuBCDA. The substrate specificity of PiuBCDA is not fully established. Herein, we report the backbone 1H, 13C and 15N resonance assignments of the 31 kDa soluble, extracellular domain of the substrate binding protein PiuA in the apo form and in complex with Ga(III) and the catechol siderophore-mimic 4-LICAM. These studies provide valuable information for further functional studies of interactions with other proteins, metals, and small molecules.