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1.
Determination of Ligand Profiles for Pseudomonas aeruginosa Solute Binding Proteins.
Fernández, M, Rico-Jiménez, M, Ortega, Á, Daddaoua, A, García García, AI, Martín-Mora, D, Torres, NM, Tajuelo, A, Matilla, MA, Krell, T
International journal of molecular sciences. 2019;(20)
Abstract
Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine, or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of them. Experimentation revealed that PA0222 was specific for γ-aminobutyrate (GABA), DppA2 for tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine. Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate, whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be predicted with confidence using bioinformatic methods, but experimental work is necessary to identify the precise ligand profile.
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2.
NMR experiments redefine the hemoglobin binding properties of bacterial NEAr-iron Transporter domains.
Macdonald, R, Mahoney, BJ, Ellis-Guardiola, K, Maresso, A, Clubb, RT
Protein science : a publication of the Protein Society. 2019;(8):1513-1523
Abstract
Iron is a versatile metal cofactor that is used in a wide range of essential cellular processes. During infections, many bacterial pathogens acquire iron from human hemoglobin (Hb), which contains the majority of the body's total iron content in the form of heme (iron protoporphyrin IX). Clinically important Gram-positive bacterial pathogens scavenge heme using an array of secreted and cell-wall-associated receptors that contain NEAr-iron Transporter (NEAT) domains. Experimentally defining the Hb binding properties of NEAT domains has been challenging, limiting our understanding of their function in heme uptake. Here we show that solution-state NMR spectroscopy is a powerful tool to define the Hb binding properties of NEAT domains. The utility of this method is demonstrated using the NEAT domains from Bacillus anthracis and Listeria monocytogenes. Our results are compatible with the existence of at least two types of NEAT domains that are capable of interacting with either Hb or heme. These binding properties can be predicted from their primary sequences, with Hb- and heme-binding NEAT domains being distinguished by the presence of (F/Y)YH(Y/F) and S/YXXXY motifs, respectively. The results of this work should enable the functions of a wide range of NEAT domain containing proteins in pathogenic bacteria to be reliably predicted.
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3.
Investigating the assembly of the bacterial type III secretion system injectisome by in vivo photocrosslinking.
Singh, N, Wagner, S
International journal of medical microbiology : IJMM. 2019;(6):151331
Abstract
Virulence-associated type III secretion systems serve the injection of bacterial effector proteins into eukaryotic host cells. These effector proteins modulate host cell biology in order to promote colonization and infection, hence type III secretion systems are often essential bacterial pathogenicity factors. The core of type III secretion systems is a cell envelope-spanning macromolecular machine called injectisome. It consists of almost twenty different components in a stoichiometry of one to more than one hundred. Assembly of this 6 MDa complex requires the coordinated integration of components from the cytoplasm, the inner membrane, the periplasm, the outer membrane and even the extracellular space of Gram-negative bacteria. Here, we review injectisome assembly with an emphasis on the techniques that were employed towards its investigation. In particular, we focus on in vivo photocrosslinking, a technique that exploits the encoding of the artificial UV-inducible crosslinking amino acid p-benzoyl-phenylalanine to identify protein-protein interactions and to delineate assembly pathways.
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4.
Creation of thermostable l-tryptophan dehydrogenase by protein engineering and its application for l-tryptophan quantification.
Matsui, D, Asano, Y
Analytical biochemistry. 2019;:57-63
Abstract
l-Tryptophan dehydrogenase is a new NAD+-dependent amino acid dehydrogenase discovered in Nostoc punctiforme. The enzyme is involved in scytonemin biosynthesis and is highly selective toward l-tryptophan. By a growth-dependent molecular evolution technique, a thermostable mutant enzyme was selected successfully. l-Tryptophan concentration in human plasma was successfully determined using the thermostable mutant of l-tryptophan dehydrogenase.
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5.
Azotobacters as biofertilizer.
Das, HK
Advances in applied microbiology. 2019;:1-43
Abstract
Azotobacters have been used as biofertilizer since more than a century. Azotobacters fix nitrogen aerobically, elaborate plant hormones, solubilize phosphates and also suppress phytopathogens or reduce their deleterious effect. Application of wild type Azotobacters results in better yield of cereals like corn, wheat, oat, barley, rice, pearl millet and sorghum, of oil seeds like mustard and sunflower, of vegetable crops like tomato, eggplant, carrot, chillies, onion, potato, beans and sugar beet, of fruits like mango and sugar cane, of fiber crops like jute and cotton and of tree like oak. In addition to the structural genes of the enzyme nitrogenase and of other accessory proteins, A. vinelandii chromosomes contain the regulatory genes nifL and nifA. NifA must bind upstream of the promoters of all nif operons for enabling their expression. NifL on activation by oxygen or ammonium, interacts with NifA and neutralizes it. Nitrogen fixation has been enhanced by deletion of nifL and by bringing nifA under the control of a constitutive promoter, resulting in a strain that continues to fix nitrogen in presence of urea fertilizer. Additional copies of nifH (the gene for the Fe-protein of nitrogenase) have been introduced into A. vinelandii, thereby augmenting nitrogen fixation. The urease gene complex ureABC has been deleted, the ammonia transport gene amtB has been disrupted and the expression of the glutamine synthase gene has been regulated to enhance urea and ammonia excretion. Gluconic acid has been produced by introducing the glucose dehydrogenase gene, resulting in enhanced solubilization of phosphate.
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6.
Life inside and out: making and breaking protein disulfide bonds in Chlamydia.
Christensen, S, McMahon, RM, Martin, JL, Huston, WM
Critical reviews in microbiology. 2019;(1):33-50
Abstract
Disulphide bonds are widely used among all domains of life to provide structural stability to proteins and to regulate enzyme activity. Chlamydia spp. are obligate intracellular bacteria that are especially dependent on the formation and degradation of protein disulphide bonds. Members of the genus Chlamydia have a unique biphasic developmental cycle alternating between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body. The proteins in the envelope of the EB are heavily cross-linked with disulphides and this is known to be critical for this infectious phase. In this review, we provide a comprehensive summary of what is known about the redox state of chlamydial envelope proteins throughout the developmental cycle. We focus especially on the factors responsible for degradation and formation of disulphide bonds in Chlamydia and how this system compares with redox regulation in other organisms. Focussing on the unique biology of Chlamydia enables us to provide important insights into how specialized suites of disulphide bond (Dsb) proteins cater for specific bacterial environments and lifecycles.
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7.
Photoactive yellow protein and its chemical probes: an approach to protein labelling in living cells.
Kumar, N, Hori, Y, Kikuchi, K
Journal of biochemistry. 2019;(2):121-127
Abstract
Labelling technologies developed over the past few years have changed the way of looking at biomolecules and have made a considerable contribution to our understanding of the functions and regulation of dynamic biological processes. One of the robust technologies employed to image proteins in a cellular environment is based on the use of chemical tags and their fluorescent probes, which provides flexibility in developing probes with a wide range of synthetic fluorophores. A variety of chemical tags, ranging from short amino acid sequences to small proteins, have been employed to generate protein-labelling systems. One such chemical tag is the photoactive yellow protein (PYP)-tag, which is a small bacterial protein, developed for the selective labelling and imaging of proteins. Herein, we briefly discuss the protein-labelling system developed based on PYP-tag technology, with a focus on the design strategy for PYP-tag labelling probes and their applications in protein imaging.
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8.
The oligopeptide ABC-importers are essential communication channels in Gram-positive bacteria.
Slamti, L, Lereclus, D
Research in microbiology. 2019;(8):338-344
Abstract
The transport of peptides in microorganisms plays an important role in their physiology and behavior, both as a nutrient source and as a proxy to sense their environment. This latter function is evidenced in Gram-positive bacteria where cell-cell communication is mediated by small peptides. Here, we highlight the importance of the oligopeptide permease (Opp) systems in the various major processes controlled by signaling peptides, such as sporulation, virulence and conjugation. We underline that the functioning of these communication systems is tightly linked to the developmental status of the bacteria via the regulation of opp gene expression by transition phase regulators.
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9.
Fast NMR method to probe solvent accessibility and disordered regions in proteins.
Faustino, AF, Barbosa, GM, Silva, M, Castanho, MARB, Da Poian, AT, Cabrita, EJ, Santos, NC, Almeida, FCL, Martins, IC
Scientific reports. 2019;(1):1647
Abstract
Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.
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10.
Transition metal transporters in rhizobia: tuning the inorganic micronutrient requirements to different living styles.
Abreu, I, Mihelj, P, Raimunda, D
Metallomics : integrated biometal science. 2019;(4):735-755
Abstract
A group of bacteria known as rhizobia are key players in symbiotic nitrogen fixation (SNF) in partnership with legumes. After a molecular exchange, the bacteria end surrounded by a plant membrane forming symbiosomes, organelle-like structures, where they differentiate to bacteroids and fix nitrogen. This symbiotic process is highly dependent on dynamic nutrient exchanges between the partners. Among these are transition metals (TM) participating as inorganic and organic cofactors of fundamental enzymes. While the understanding of how plant transporters facilitate TMs to the very near environment of the bacteroid is expanding, our knowledge on how bacteroid transporters integrate to TM homeostasis mechanisms in the plant host is still limited. This is significantly relevant considering the low solubility and scarcity of TMs in soils, and the in crescendo gradient of TM bioavailability rhizobia faces during the infection and bacteroid differentiation processes. In the present work, we review the main metal transporter families found in rhizobia, their role in free-living conditions and, when known, in symbiosis. We focus on discussing those transporters which could play a significant role in TM-dependent biochemical and physiological processes in the bacteroid, thus paving the way towards an optimized SNF.