A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria.

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3-4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A-(GlcNAc)2 complex in a 'half-open' conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane-transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.

Comparative Integrated Omics Analysis of the Hfq Regulon in Bordetella pertussis.

Bordetella pertussis is a Gram-negative strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Previously, we have shown that RNA chaperone Hfq is required for virulence of B. pertussis. Furthermore, microarray analysis revealed that a large number of genes are affected by the lack of Hfq. This study represents the first attempt to characterize the Hfq regulon in bacterial pathogen using an integrative omics approach. Gene expression profiles were analyzed by RNA-seq and protein amounts in cell-associated and cell-free fractions were determined by LC-MS/MS technique. Comparative analysis of transcriptomic and proteomic data revealed solid correlation (r2 = 0.4) considering the role of Hfq in post-transcriptional control of gene expression. Importantly, our study confirms and further enlightens the role of Hfq in pathogenicity of B. pertussis as it shows that Δhfq strain displays strongly impaired secretion of substrates of Type III secretion system (T3SS) and substantially reduced resistance to serum killing. On the other hand, significantly increased production of proteins implicated in transport of important metabolites and essential nutrients observed in the mutant seems to compensate for the physiological defect introduced by the deletion of the hfq gene.

Real-time, label-free characterization of oligosaccharide-binding proteins using carbohydrate microarrays and an ellipsometry-based biosensor.

Carbohydrates present on cell surfaces mediate cell behavior through interactions with other biomolecules. Due to their structural complexity, diversity, and heterogeneity, it is difficult to fully characterize a variety of carbohydrates and their binding partners. As a result, novel technologies for glycomics applications have been developed, including carbohydrate microarrays and label-free detection methods. In this paper, we report using the combination of oligosaccharide microarrays and the label-free oblique-incidence reflectivity difference (OI-RD) microscopy for real-time characterization of oligosaccharide binding proteins. Aminated human milk oligosaccharides were immobilized on epoxy-coated glass substrates as microarrays for reactions with Family 1 of solute binding proteins from Bifidobacterium longum subsp. infantis (B. infantis). Binding affinities of these protein-oligosaccharide interactions showed preferences of Family 1 of solute binding proteins to host glycans, which helps in characterizing the complex process of human milk oligosaccharides foraging by B. infantis.

Exploring solute binding proteins in Pseudomonas aeruginosa that bind to γ-aminobutyrate and 5-aminovalerate and their role in activating sensor kinases.

The standard method of receptor activation involves the binding of signals or signal-loaded solute binding proteins (SBPs) to sensor domains. Many sensor histidine kinases (SHKs), which are activated by SBP binding, are encoded adjacent to their corresponding sbp gene. We examined three SBPs of Pseudomonas aeruginosa PAO1, encoded near the genes for the AgtS (PA0600) and AruS (PA4982) SHKs, to determine how common this arrangement is. Ligand screening and microcalorimetric studies revealed that the SBPs PA0602 and PA4985 preferentially bind to GABA (KD = 2.3 and 0.58 µM, respectively), followed by 5-aminovalerate (KD = 30 and 1.6 µM, respectively) and ethanoldiamine (KD = 2.3 and 0.58 µM, respectively). In contrast, AgtB (PA0604) exclusively recognizes 5-aminovaleric acid (KD = 2.9 µM). However, microcalorimetric titrations did not show any binding between the AgtS sensor domain and AgtB or PA0602, regardless of the presence of ligands. Similarly, bacterial two-hybrid assays did not demonstrate an interaction between PA4985 and the AruS sensor domain. Therefore, sbp and shk genes located nearby are not always functionally linked. We previously identified PA0222 as a GABA-specific SBP. The presence of three SBPs for GABA may be linked to GABA's role as a trigger for P. aeruginosa virulence.

S100A12 promotes Mn(II) binding to pneumococcal PsaA and staphylococcal MntC by Zn(II) sequestration.

Human S100A12 (calgranulin C, EN-RAGE) is a Zn(II)-sequestering host-defense protein that contributes to the metal-withholding innate immune response against microbial pathogens. S100A12 coordinates Zn(II) ions at two His3Asp sites with high affinity. A similar His3Asp site found in calprotectin (S100A8/S100A9, calgranulin A/B), a closely related human S100 protein, can sequester divalent metal ions from the solute-binding proteins (SBPs) pneumococcal PsaA (pneumococcal surface protein A) and staphylococcal MntC (manganese transport protein C). Both SBPs are components of Mn(II) transporters and capture extracellular Mn(II) ions for subsequent delivery into the bacterial cytosol. Nevertheless, PsaA and MntC exhibit a thermodynamic preference for Zn(II) over Mn(II), and Zn(II) binding can interfere with Mn(II) acquisition. In this work, we have used a biotinylated variant of S100A12 to show that S100A12 can sequester Zn(II) ions from PsaA and MntC. Moreover, electron paramagnetic resonance (EPR) spectroscopy indicates that by sequestering Zn(II) from Zn(II)-bound PsaA and MntC, S100A12 promotes Mn(II) binding to the SBPs. These results inform the function of S100A12 in Zn(II) sequestration, and further suggest that Zn(II)-sequestering S100 proteins may inadvertently protect bacterial pathogens during infection.

Conformational flexibility in the zinc solute-binding protein ZnuA.

Zinc is an essential metal for all kingdoms of life, making its transport across the cell membrane a critical function. In bacteria, high-affinity zinc import is accomplished by ATP-binding cassette (ABC) transporters, which rely on extracellular solute-binding proteins (SBPs) of cluster A-I to acquire the metal and deliver it to the membrane permease. These systems are important for survival and virulence, making them attractive targets for the development of novel antibiotics. Citrobacter koseri is an emerging pathogen with extensive antibiotic resistance. High-affinity zinc binding to the C. koseri cluster A-I SBP ZnuA has been characterized and the structure of the zinc-bound (holo) form has been determined by X-ray crystallography. Remarkably, despite 95% sequence identity to the ZnuA homologue from Salmonella enterica, C. koseri ZnuA exhibits a different zinc-coordination environment and a closed rather than an open conformation. Comparison with structures of another close ZnuA homologue from Escherichia coli suggests a surprisingly flexible conformational landscape that may be important for efficient zinc binding and/or delivery to the membrane permease.

The Repertoire of Solute-Binding Proteins of Model Bacteria Reveals Large Differences in Number, Type, and Ligand Range.

Solute-binding proteins (SBPs) are of central physiological relevance for bacteria. They are located in the extracytosolic space, where they present substrates to transporters but also stimulate different types of transmembrane receptors coordinating compound uptake with signal transduction. SBPs are a superfamily composed of proteins recognized by 45 Pfam profiles. The definition of SBP profiles for bacteria is hampered by the fact that these Pfam profiles recognize sensor domains for different types of signaling proteins or cytosolic proteins with alternative functions. We report here the retrieval of the SBPs from 49 bacterial model strains with different lifestyles and phylogenetic distributions. Proteins were manually curated, and the ligands recognized were predicted bioinformatically. There were very large differences in the number and type of SBPs between strains, ranging from 7 SBPs in Helicobacter pylori 26695 to 189 SBPs in Sinorhizobium meliloti 1021. SBPs were found to represent 0.22 to 5.13% of the total protein-encoding genes. The abundance of SBPs was largely determined by strain phylogeny, and no obvious link with the bacterial lifestyle was noted. Most abundant (36%) were SBPs predicted to recognize amino acids or peptides, followed by those expected to bind different sugars (18%). To the best of our knowledge, this is the first comparative study of bacterial SBP repertoires. Given the importance of SBPs in nutrient uptake and signaling, this study enhances the knowledge of model bacteria and will permit the definition of SBP profiles of other strains. IMPORTANCE SBPs are essential components for many transporters, but multiple pieces of more recent evidence indicate that the SBP-mediated stimulation of different transmembrane receptors is a general and widespread signal transduction mechanism in bacteria. The double function of SBPs in coordinating transport with signal transduction remains to a large degree unexplored and represents a major research need. The definition of the SBP repertoire of the 49 bacterial model strains examined here, along with information on their cognate ligand profiles forms the basis to close this gap in knowledge. Furthermore, this study provides information on the forces that have driven the evolution of transporters with different ligand specificities in bacteria that differ in phylogenetics and lifestyle. This article is also a first step in setting up automatic algorithms that permit the large-scale identification of the SBP repertoire in proteomes.

Periplasmic solute-binding proteins: Structure classification and chitooligosaccharide recognition.

Periplasmic solute-binding proteins (SBPs) serve as molecular shuttles that assist the transport of small solutes from the outer membrane to the inner membrane of all Gram-negative bacteria. Based on the available crystal structures, SBPs are classified into seven clusters, A-G, and are further divided into subclusters, IV. This minireview is focused on the classification, structure and substrate specificity of a distinct class of SBPs specific for chitooligosaccharides (CBPs). To date, only two structures of CBP homologues, VhCBP and VcCBP, have been reported in the marine Vibrio species, with exposition of their limited function. The Vibrio CBPs are structurally classified as cluster C/subcluster IV SBPs that exclusively recognize ß-1,4- or ß-1,3-linked linear oligosaccharides. The overall structural feature of the Vibrios CBPs is most similar to the cellobiose-binding orthologue from the hyperthermophilic bacterium Thermotoga maritima. This similarity provides an opportunity to engineer the substrate specificity of the proteins and to control the uptake of chitinous and cellulosic nutrients in marine bacteria.

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

BACKGROUND: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. RESULTS: Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. CONCLUSIONS: This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.