scholarly journals Actinobacillus utilizes a binding-protein dependent ABC transporter to acquire Vitamin B6

2020 ◽  
Author(s):  
Chuxi Pan ◽  
Alexandra Zimmer ◽  
Megha Shah ◽  
Minhsang Huynh ◽  
Christine C.L. Lai ◽  
...  
Keyword(s):  
Electron Density ◽  
High Efficiency ◽  
Binding Protein ◽  
Titration Calorimetry ◽  
Vitamin B ◽  
Uptake System ◽  
Periplasmic Binding Protein ◽  
Resolution Electron ◽  
Key Enzyme ◽  
K 12

AbstractBacteria require high efficiency uptake systems to survive and proliferate in nutrient limiting environments, such as those found in the host. The ABC transporters at the bacterial plasma membrane provide a mechanism for transport of many substrates. We recently demonstrated that an AfuABC operon, previously annotated as encoding a ferrous iron uptake system, is in fact a cyclic hexose/heptose-phosphate transporter with high selectivity and specificity for these metabolites. In this study, we examine a second operon containing a periplasmic binding protein discovered in Actinobacillus for its potential role in nutrient acquisition. Using electron density obtained from the crystal structure of the periplasmic binding protein we modeled a pyridoxal-5’-phosphate (P5P/PLP/Vitamin B6) ligand into the atomic resolution electron density map. The identity of the Vitamin B6 bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from A. pleuropneumoniae into an E. coli K-12 strain that was devoid of a key enzyme required for Vitamin B6 synthesis. The growth of this strain at low levels of Vitamin B6 supports the role of this newly identify operon in Vitamin B6 uptake.

scholarly journals Characterization of a Sinorhizobium melilotiATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake

2000 ◽  
Vol 182 (13) ◽  
pp. 3717-3725 ◽  
Author(s):  
Eric Boncompagni ◽  
Laurence Dupont ◽  
Tam Mignot ◽  
Magne Østeräs ◽  
Annie Lambert ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Binding Protein ◽  
Compatible Solutes ◽  
Site Directed Mutagenesis ◽  
Uptake System ◽  
Periplasmic Binding Protein ◽  
Gene Encoding ◽  
Inhibition Of Growth

ABSTRACT The symbiotic soil bacterium Sinorhizobium melilotiuses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli(ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed inhut mutants (hutX and hutH2). Expression analysis of the hut operon determined using ahutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

scholarly journals Znu Is the Predominant Zinc Importer in Yersinia pestis during In Vitro Growth but Is Not Essential for Virulence

2010 ◽  
Vol 78 (12) ◽  
pp. 5163-5177 ◽  
Author(s):  
Daniel C. Desrosiers ◽  
Scott W. Bearden ◽  
Ildefonso Mier ◽  
Jennifer Abney ◽  
James T. Paulley ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Binding Protein ◽  
Bacterial Pathogens ◽  
Titration Calorimetry ◽  
Growth Defect ◽  
Uptake System ◽  
High Affinity ◽  
Zn Uptake ◽  
Low Concentrations

ABSTRACT Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacterium's Zn requirements under in vivo metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.

scholarly journals Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System

1998 ◽  
Vol 180 (18) ◽  
pp. 4828-4833 ◽  
Author(s):  
Roberto T. F. Celis ◽  
Peter F. Leadlay ◽  
Ipsita Roy ◽  
Anne Hansen
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Transport System ◽  
Binding Proteins ◽  
Binding Protein ◽  
Transport Systems ◽  
Periplasmic Binding Protein ◽  
Transport Atpase ◽  
Periplasmic Binding Proteins ◽  
K 12

ABSTRACT In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.

scholarly journals The Periplasmic Cyclodextrin Binding Protein CymE from Klebsiella oxytoca and Its Role in Maltodextrin and Cyclodextrin Transport

1998 ◽  
Vol 180 (10) ◽  
pp. 2630-2635 ◽  
Author(s):  
Markus Pajatsch ◽  
Maria Gerhart ◽  
Ralf Peist ◽  
Reinhold Horlacher ◽  
Winfried Boos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Quenching ◽  
Dissociation Constants ◽  
Binding Protein ◽  
Klebsiella Oxytoca ◽  
Uptake System ◽  
Gene Products ◽  
Periplasmic Binding Protein ◽  
E Coli

ABSTRACT Klebsiella oxytoca M5a1 has the capacity to transport and to metabolize α-, β- and γ-cyclodextrins. Cyclodextrin transport is mediated by the products of the cymE,cymF, cymG, cymD, andcymA genes, which are functionally homologous to themalE, malF, malG, malK, and lamB gene products of Escherichia coli. CymE, which is the periplasmic binding protein, has been overproduced and purified. By substrate-induced fluorescence quenching, the binding of ligands was analyzed. CymE bound α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, with dissociation constants (Kd ) of 0.02, 0.14 and 0.30 μM, respectively, and linear maltoheptaose, with a Kd of 70 μM. In transport experiments, α-cyclodextrin was taken up by thecym system of K. oxytoca three to five times less efficiently than maltohexaose by the E. coli maltose system. Besides α-cyclodextrin, maltohexaose was also taken up by theK. oxytoca cym system, but because of the inability of maltodextrins to induce the cym system, growth of E. coli mal mutants on linear maltodextrin was not observed when the cells harbored only the cym uptake system. Strains which gained this capacity by mutation could easily be selected, however.

scholarly journals Periplasmic binding protein dependent transport system for maltose and maltodextrins: some recent studies

1989 ◽  
Vol 63 (1-2) ◽  
pp. 53-60
Author(s):  
W. Saurin ◽  
E. Francoz ◽  
P. Martineau ◽  
A. Charbit ◽  
E. Dassa ◽  
...  
Keyword(s):  
Transport System ◽  
Binding Protein ◽  
Periplasmic Binding Protein ◽  
Dependent Transport

A Comparison of Two Algorithms for Electron-Density Map Improvement by Introduction of Atomicity: Skeletonization, and Map Sorting Followed by Refinement

1998 ◽  
Vol 54 (1) ◽  
pp. 81-85 ◽  
Author(s):  
F. M. D. Vellieux
Keyword(s):  
Electron Density ◽  
Initial Density ◽  
Acta Cryst ◽  
Density Maps ◽  
Resolution Electron ◽  
Crystallographic Symmetry ◽  
Density Map ◽  
Ornithine Transcarbamoylase ◽  
Electron Density Map ◽  
Pseudo Atom

A comparison has been made of two methods for electron-density map improvement by the introduction of atomicity, namely the iterative skeletonization procedure of the CCP4 program DM [Cowtan & Main (1993). Acta Cryst. D49, 148–157] and the pseudo-atom introduction followed by the refinement protocol in the program suite DEMON/ANGEL [Vellieux, Hunt, Roy & Read (1995). J. Appl. Cryst. 28, 347–351]. Tests carried out using the 3.0 Å resolution electron density resulting from iterative 12-fold non-crystallographic symmetry averaging and solvent flattening for the Pseudomonas aeruginosa ornithine transcarbamoylase [Villeret, Tricot, Stalon & Dideberg (1995). Proc. Natl Acad. Sci. USA, 92, 10762–10766] indicate that pseudo-atom introduction followed by refinement performs much better than iterative skeletonization: with the former method, a phase improvement of 15.3° is obtained with respect to the initial density modification phases. With iterative skeletonization a phase degradation of 0.4° is obtained. Consequently, the electron-density maps obtained using pseudo-atom phases or pseudo-atom phases combined with density-modification phases are much easier to interpret. These tests also show that for ornithine transcarbamoylase, where 12-fold non-crystallographic symmetry is present in the P1 crystals, G-function coupling leads to the simultaneous decrease of the conventional R factor and of the free R factor, a phenomenon which is not observed when non-crystallographic symmetry is absent from the crystal. The method is far less effective in such a case, and the results obtained suggest that the map sorting followed by refinement stage should be by-passed to obtain interpretable electron-density distributions.

Detailed analysis of the atrial natriuretic factor receptor hormone-binding domain crystal structure

2001 ◽  
Vol 79 (8) ◽  
pp. 692-704 ◽  
Author(s):  
Focco van den Akker
Keyword(s):  
Crystal Structure ◽  
Atrial Natriuretic Factor ◽  
Binding Protein ◽  
Chloride Ion ◽  
Binding Domain ◽  
Protein Fold ◽  
Hormone Binding ◽  
Periplasmic Binding Protein ◽  
Natriuretic Factor ◽  
Hormone Binding Domain

The X-ray crystal structure of the dimerized atrial natriuretic factor (ANF) receptor hormone-binding domain has provided a first structural view of this anti-hypertensive receptor. The structure reveals a surprising evolutionary link to the periplasmic-binding protein fold family. Furthermore, the presence of a chloride ion in the membrane distal domain and the presence of a second putative effector pocket suggests that the extracellular domain of this receptor is allosterically regulated. The scope of this article is to extensively review the data published on this receptor and to correlate it with the hormone-binding domain structure. In addition, a more detailed description is provided of the important features of this structure including the different binding sites for the ANF hormone, chloride ion, putative effector pocket, glycosylation sites, and dimer interface.Key words: crystal structure, periplasmic-binding protein fold, guanylyl cyclase, hormone receptor.

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