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.

Hydrogenotrophic Methanogenesis Under Alkaline Conditions

A cement-based geological disposal facility (GDF) is one potential option for the disposal of intermediate level radioactive wastes. The presence of both organic and metallic materials within a GDF provides the opportunity for both acetoclastic and hydrogenotrophic methanogenesis. However, for these processes to proceed, they need to adapt to the alkaline environment generated by the cementitious materials employed in backfilling and construction. Within the present study, a range of alkaline and neutral pH sediments were investigated to determine the upper pH limit and the preferred route of methane generation. In all cases, the acetoclastic route did not proceed above pH 9.0, and the hydrogenotrophic route dominated methane generation under alkaline conditions. In some alkaline sediments, acetate metabolism was coupled to hydrogenotrophic methanogenesis via syntrophic acetate oxidation, which was confirmed through inhibition studies employing fluoromethane. The absence of acetoclastic methanogenesis at alkaline pH values (>pH 9.0) is attributed to the dominance of the acetate anion over the uncharged, undissociated acid. Under these conditions, acetoclastic methanogens require an active transport system to access their substrate. The data indicate that hydrogenotrophic methanogenesis is the dominant methanogenic pathway under alkaline conditions (>pH 9.0).

Methotrexate—Mechanisms of Drug Action

Methotrexate (MTX), a structural analogue of folic acid, that inhibits cell division (mainly in the S phase of the cell cycle) is commonly used for the treatment of many cancers as well for severe and resistant forms of autoimmune pathologies and inflammatory disorders. This paragraph of clinical overview presents state of knowledge with regards to different pathways of MTX active transport system, mechanisms of action and its applications as immunosuppressive drug and anticancer agent.  

Uptake of L-Alanine and Its Distinct Roles in the Bioenergetics of Trypanosoma cruzi

ABSTRACT Amino acids participate in several critical processes in the biology of trypanosomatids, such as osmoregulation, cell differentiation, and host cell invasion. Some of them provide reducing power for mitochondrial ATP synthesis. It was previously shown that alanine, which is formed mainly by the amination of pyruvate, is a metabolic end product formed when parasites are replicating in a medium rich in glucose and amino acids. It was shown as well that this amino acid can also be used for the regulation of cell volume and resistance to osmotic stress. In this work, we demonstrate that, despite it being an end product of its metabolism, Trypanosoma cruzi can take up and metabolize l-Ala through a low-specificity nonstereoselective active transport system. The uptake was dependent on the temperature in the range between 10 and 40°C, which allowed us to calculate an activation energy of 66.4 kJ/mol and estimate the number of transporters per cell at ~436,000. We show as well that, once taken up by the cells, l-Ala can be completely oxidized to CO2, supplying electrons to the electron transport chain, maintaining the electrochemical proton gradient across the mitochondrial inner membrane, and supporting ATP synthesis in T. cruzi epimastigotes. Our data demonstrate a dual role for Ala in the parasite’s bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism. IMPORTANCE It is well known that trypanosomatids such as the etiological agent of Chagas’ disease, Trypanosoma cruzi, produce alanine as a main end product of their energy metabolism when they grow in a medium containing glucose and amino acids. In this work, we investigated if under starvation conditions (which happen during the parasite life cycle) the secreted alanine could be recovered from the extracellular medium and used as an energy source. Herein we show that indeed, in parasites submitted to metabolic stress, this metabolite can be taken up and used as an energy source for ATP synthesis, allowing the parasite to extend its survival under starvation conditions. The obtained results point to a dual role for Ala in the parasite’s bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism.

Identification and Regulation of Genes for Cobalamin Transport in the Cyanobacterium Synechococcus sp. Strain PCC 7002

ABSTRACTThe cyanobacteriumSynechococcussp. strain PCC 7002 is a cobalamin auxotroph and utilizes this coenzyme solely for the synthesis ofl-methionine by methionine synthase (MetH).Synechococcussp. strain PCC 7002 is unable to synthesize cobalaminde novo, and because of the large size of this tetrapyrrole, an active-transport system must exist for cobalamin uptake. Surprisingly, no cobalamin transport system was identified in the initial annotation of the genome of this organism. With more sophisticatedin silicoprediction tools, abtuB-cpdA-btuC-btuFoperon encoding components putatively required for a B12uptake (btu) system was identified. The expression of these genes was predicted to be controlled by a cobalamin riboswitch. Global transcriptional profiling by high-throughput RNA sequencing of a cobalamin-independent form ofSynechococcussp. strain PCC 7002 grown in the absence or presence of cobalamin confirmed regulation of thebtuoperon by cobalamin. Pérez et al. (A. A. Pérez, Z. Liu, D. A. Rodionov, Z. Li, and D. A. Bryant, J Bacteriol 198:2743–2752, 2016,http://dx.doi.org/10.1128/JB.00475-16) developed a cobalamin-dependent yellow fluorescent protein reporter system in aSynechococcussp. strain PCC 7002 variant that had been genetically modified to allow cobalamin-independent growth. This reporter system was exploited to validate components of thebtuuptake system by assessing the ability of targeted mutants to transport cobalamin. ThebtuBpromoter and a variant counterpart mutated in an essential element of the predicted cobalamin riboswitch were fused to ayfpreporter. The combined data indicate that thebtuB-cpdA-btuF-btuCoperon in this cyanobacterium is transcriptionally regulated by a cobalamin riboswitch.IMPORTANCEWith a cobalamin-regulated reporter system for expression of yellow fluorescent protein, genes previously misidentified as encoding subunits of a siderophore transporter were shown to encode components of cobalamin uptake in the cyanobacteriumSynechococcussp. strain PCC 7002. This study demonstrates the importance of experimental validation ofin silicopredictions and provides a general scheme forin vivoverification of similar cobalamin transport systems. A putative cobalamin riboswitch was identified inSynechococcussp. strain PCC 7002. This riboswitch acts as a potential transcriptional attenuator of thebtuoperon that encodes the components of the cobalamin active-transport system.

The reference range of serum magnesium substance concentration among healthy young adults at Makerere University College of Health Sciences 2012

Background: Magnesium is the second most abundant intracellular cation, with only a small proportion of the body’s content being in the extracellular fluid. It is required for the active transport of other cations such as calcium, sodium and potassium across the membrane by active transport system. It is also needed for many intracellular metabolic pathways. This study was carried to establish the reference intervals for serum magnesium substance concentration among healthy medical students in Uganda.Methods: This was purposive study in which ante-cubital venous blood samples were drawn without stasis from 60 healthy, natively Ugandan pre-clinical medical students and analysed without delay using Cobasintegra 400/700/800 automated analyser which flagged each result using the in-built seemingly temperate reference range of 0.65-1.05 mmol/L.Results: The distribution of serum magnesium substance concentration was unimodal, leptokurtic, and positively skewed with empirical range of 0.86 – 1.32 mmol/L. There was no result flagged as low. Twenty-six out of sixty (43.3%) results were flagged as high values while none approached 2.0 mmol/L, considered the threshold of hypermagnesaemia symptoms. Using the central 95 percentile, the reference range was set as 0.81 – 1.29 mmol/L which is higher and slightly broader than the 0.65 – 1.05 mmol/L often quoted for populations in temperate regions and in-built in automated analysers exported even to the tropics.Conclusion: Reference ranges were higher in the studied healthy young adults in Uganda than those in the temperate regions. Effort should therefore be made to enable our laboratories establish their own reference values.

Clarithromycin Transport by Gingival Fibroblasts and Epithelial Cells

Macrolide antibiotics penetrate cells, but the mechanism by which this occurs is unclear. The objective of this study was to characterize the mechanisms of clarithromycin uptake by gingival fibroblasts and oral epithelium. Cultured human gingival fibroblasts and SCC-25 cells were incubated with [3H]-clarithromycin. We assayed clarithromycin transport by measuring cell-associated radioactivity over time. Fibroblasts and epithelial cells rapidly accumulated clarithromycin, attaining steady-state intracellular concentrations within 15 minutes. Incubation in medium containing 2 μg/mL clarithromycin yielded steady-state intracellular concentrations of 75.8 μg/mL in fibroblasts and 6.6 μg/mL in SCC-25 cells. Clarithromycin transport exhibited Michaelis-Menten kinetics and was inhibited below 37°C. The Michaelis constants for fibro-blasts and SCC-25 cells were 78.4 and 227 μg/mL, respectively, while the maximum transport velocities were 264 and 381 ng/min/106 cells, respectively. Thus, both types of cells take up clarithromycin via a concentrative active transport system. By increasing intracellular clarithromycin levels, this system may enhance the effectiveness of clarithromycin against invasive periodontal pathogens.

Access of Extracellular Cations to their Binding Sites in Na,K-ATPase: Role of the Second Extracellular Loop of the α Subunit

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na+ and K+ gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na+ ions and releases them on the extracellular side of the membrane, and then binds two extracellular K+ ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the α subunit of Na,K-ATPase are known to be involved in Na+ and K+ binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat α1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K+, and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K+ to its binding site.