The Xanthomonas citri pv. citri (X. citri) is a phytopathogenic bacterium that infects different species of citrus plants where it causes canker disease. The adaptation to different habitats is related to the ability of the cells to metabolize and to assimilate diverse compounds, including sulfur, an essential element for all organisms. In Escherichia coli, the necessary sulfur can be obtained by a set of proteins whose genes belong to the cys regulon. Although the cys regulon proteins and their importance have been described in many other bacteria, there are no data related to these proteins in X. citri or in the Xanthomonas genus. The study of the relevance of these systems in these phytopathogenic bacteria that have distinct mechanisms of infection is one essential step toward understanding their physiology. In this work, we used bioinformatics, molecular modeling and transcription analysis (RT-PCR) to identify and characterize the putative cys regulon genes in X. citri.

Transmembrane receptors are integral components of sensory pathways in prokaryotes. These receptors share a common dimeric architecture, consisting in its basic form of an N-terminal extracellular sensor, transmembrane helices, and an intracellular effector. As an exception, we have identified an archaeal receptor family--exemplified by Af1503 from Archaeoglobus fulgidus--that is C-terminally shortened, lacking a recognizable effector module. Instead, a HAMP domain forms the sole extension for signal transduction in the cytosol. Here, we examine the gene environment of Af1503-like receptors and find a frequent association with transmembrane transport proteins. Furthermore, we identify and define a closely associated new protein domain family, which we characterize structurally using Af1502 from A. fulgidus. Members of this family are found both as stand-alone proteins and as domains within extant receptors. In general, the latter appear as connectors between the solute carrier 5 (SLC5)-like transmembrane domains and two-component signal transduction (TCST) domains. This is seen, for example, in the histidine kinase CbrA, which is a global regulator of metabolism, virulence, and antibiotic resistance in Pseudomonads. We propose that this newly identified domain family mediates signal transduction in systems regulating transport processes and name it STAC, for SLC and TCST-Associated Component.

The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell movement. Bioinformatics analysis identified two hydrophilic non-contiguous regions (R1 and R2) rich in the basic amino acids lysine and arginine and with the predicted secondary structure of an α-helix. Different approaches were used to determine the implication of the R1 and R2 regions in RNA binding, plasmodesmata (PD) targeting and cell-to-cell movement. EMSA (Electrophoretic Mobility Shift Assay) showed that both regions have RNA-binding activity whereas that mutational analysis reported that either deletion of any of these regions, or loss of the basic amino acids, interfered with the viral intercellular movement. Subcellular localization studies showed that PMoV MP locates at PD. Mutants designed to impeded cell-to-cell movement failed to accumulate at PD indicating that basic residues in both R1 and R2 are critical for binding the MP at PD.

The United States faces a national crisis involving opioid medications, where currently more than 130 people die every day. To combat this epidemic, a better understanding is needed of how opioids penetrate into the central nervous system (CNS) to facilitate pain relief and, potentially, result in addiction and/or misuse. Animal models, however, are a poor predictor of blood-brain barrier (BBB) transport and CNS drug penetration in humans, and many traditional 2D cell culture models of the BBB and neurovascular unit have inadequate barrier function and weak or inappropriate efflux transporter expression. Here, we sought to better understand opioid transport mechanisms using a simplified microfluidic neurovascular unit (NVU) model consisting of human brain microvascular endothelial cells (BMECs) co-cultured with astrocytes.

Plasma membrane transporter SLC6A14 transports all neutral and basic amino acids in a Na/Cl - dependent way and it is up-regulated in many types of cancer. Mass spectrometry analysis of overexpressed SLC6A14-associated proteins identified, among others, the presence of cytosolic heat shock proteins (HSPs) and co-chaperones. We detected co-localization of overexpressed and native SLC6A14 with HSP90-beta and HSP70 (HSPA14). Proximity ligation assay confirmed a direct interaction of overexpressed SLC6A14 with both HSPs. Treatment with radicicol and VER155008, specific inhibitors of HSP90 and HSP70, respectively, attenuated these interactions and strongly reduced transporter presence at the cell surface, what resulted from the diminished level of the total transporter protein. Distortion of SLC6A14 proper folding by both HSPs inhibitors directed the transporter towards endoplasmic reticulum-associated degradation pathway, a process reversed by the proteasome inhibitor - bortezomib. As demonstrated in an in vitro ATPase assay of recombinant purified HSP90-beta, the peptides corresponding to C-terminal amino acid sequence following the last transmembrane domain of SLC6A14 affected the HSP90-beta activity. These results indicate that a plasma membrane protein folding can be controlled not only by chaperones in the endoplasmic reticulum, but also those localized in the cytosol.

Plants acquire nitrogen in the form of amino acids from the soil, and transport proteins located in the plasma membrane of root cells are required for this process. It was found that the Arabidopsis lysine-histidine-like transporter LHT6 is expressed in root cells important for amino acid uptake, including the epidermis, root hairs, and cortex. Transport studies with lht6 mutants using high levels of amino acids demonstrated that LHT6 is in fact involved in amino acid uptake. To determine if LHT6 plays a role in nitrogen acquisition at soil amino acid concentrations, growth and uptake studies were performed with low levels of toxic amino acid analogues and radiolabelled amino acids, respectively. In addition, mutants of AAP1, another root amino acid transporter, and lht6/aap1 double mutants were examined. The results showed that LHT6 is involved in uptake of acidic amino acids, glutamine and alanine, and probably phenylalanine. LHT6 seems not to transport basic or other neutral amino acids, or, alternatively, other transporters might compensate for eliminated LHT6 function. Previous studies suggested that AAP1 only takes up amino acids at high concentrations; however, here it is demonstrated that the transporter functions in acquisition of glutamate and neutral amino acids when present at soil concentrations. When comparing the characterized root uptake systems, it appears that transporters both with overlapping substrate specificity and with preference for specific substrates are required to access the soil amino acid pool.

Time-restricted feeding (TRF) improves metabolism independent of dietary macronutrient composition or energy restriction. To elucidate mechanisms underpinning the effects of short-term TRF, we investigated skeletal muscle and serum metabolic and transcriptomic profiles from 11 men with overweight/obesity after TRF (8 h day-1) and extended feeding (EXF, 15 h day-1) in a randomised cross-over design (trial registration: ACTRN12617000165381). Here we show that muscle core clock gene expression was similar after both interventions. TRF increases the amplitude of oscillating muscle transcripts, but not muscle or serum metabolites. In muscle, TRF induces rhythmicity of several amino acid transporter genes and metabolites. In serum, lipids are the largest class of periodic metabolites, while the majority of phase-shifted metabolites are amino acid related. In conclusion, short-term TRF in overweight men affects the rhythmicity of serum and muscle metabolites and regulates the rhythmicity of genes controlling amino acid transport, without perturbing core clock gene expression.

The sodium-dependent neutral amino acid transporter 2 (SNAT2), which has dual transport/receptor functions, is well documented in eukaryotes and some mammalian systems, but has not yet been verified in piglets. The objective of this study was to investigate the characteristics and regulation of SNAT2 in the small intestine of piglets. The 1,521-bp porcine full cDNA sequence of SNAT2 (KC769999) from the small intestine of piglets was cloned. The open reading frame of cDNA encodes 506 deduced amino acid residues with a calculated molecular mass of 56.08 kDa and an isoelectric point (pI) of 7.16. Sequence alignment and phylogenetic analysis revealed that SNAT2 is highly evolutionarily conserved in mammals. SNAT2 mRNA can be detected in the duodenum, jejunum and ileum by real-time quantitative PCR. During the suckling period from days 1 to 21, the duodenum had the highest abundance of SNAT2 mRNA among the three segments of the small intestine. There was a significant decrease in the expression of SNAT2 mRNA in the duodenal and jejunal mucosa and in the expression of SNAT2 protein in the jejunal and ileal mucosa on day 1 after weaning (P < 0.05). Studies with enterocytes in vitro showed that amino acid starvation and supplementation with glutamate, arginine or leucine enhanced, while supplementation with glutamine reduced, SNAT2 mRNA expression (P < 0.05). These results regarding the characteristics and regulation of SNAT2 should help to provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine.

Plants need to maintain a low Na+/K+ ratio for their survival and growth when there is high sodium concentration in soil. Under these circumstances, the high affinity K+ transporter (HKT) and its homologs are known to perform a critical role with HKT1;5 as a major player in maintaining Na+ concentration. Preferential expression of HKT1;5 in roots compared to shoots was observed in rice and rice-like genotypes from real time PCR, microarray, and RNAseq experiments and data. Its expression trend was generally higher under increasing salt stress in sensitive IR29, tolerant Pokkali, both glycophytes; as well as the distant wild rice halophyte, Porteresia coarctata, indicative of its importance during salt stress. These results were supported by a low Na+/K+ ratio in Pokkali, but a much lower one in P. coarctata. HKT1;5 has functional variability among salt sensitive and tolerant varieties and multiple sequence alignment of sequences of HKT1;5 from Oryza species and P. coarctata showed 4 major amino acid substitutions (140 P/A/T/I, 184 H/R, D332H, V395L), with similarity amongst the tolerant genotypes and the halophyte but in variance with sensitive ones. The best predicted 3D structure of HKT1;5 was generated using Ktrab potassium transporter as template. Among the four substitutions, conserved presence of aspartate (332) and valine (395) in opposite faces of the membrane along the Na+/K+ channel was observed only for the tolerant and halophytic genotypes. A model based on above, as well as molecular dynamics simulation study showed that valine is unable to generate strong hydrophobic network with its surroundings in comparison to leucine due to reduced side chain length. The resultant alteration in pore rigidity increases the likelihood of Na+ transport from xylem sap to parenchyma and further to soil. The model also proposes that the presence of aspartate at the 332 position possibly leads to frequent polar interactions with the extracellular loop polar residues which may shift the loop away from the opening of the constriction at the pore and therefore permit easy efflux of the Na+. These two substitutions of the HKT1;5 transporter probably help tolerant varieties maintain better Na+/K+ ratio for survival under salt stress.

Amino acid transporters (AATs) that transport amino acids across cellular membranes are essential for plant growth and development. To date, a genome-wide overview of the AAT gene family in rice is not yet available.

Proper storage of excessive dietary fat into subcutaneous adipose tissue (SAT) prevents ectopic lipid deposition-induced insulin resistance, yet the underlying mechanism remains unclear. Here, we identify angiopoietin-2 (Angpt2)-integrin α5β1 signaling as an inducer of fat uptake specifically in SAT. Adipocyte-specific deletion of Angpt2 markedly reduced fatty acid uptake and storage in SAT, leading to ectopic lipid accumulation in glucose-consuming organs including skeletal muscle and liver and to systemic insulin resistance. Mechanistically, Angpt2 activated integrin α5β1 signaling in the endothelium and triggered fatty acid transport via CD36 and FATP3 into SAT. Genetic or pharmacological inhibition of the endothelial integrin α5β1 recapitulated adipocyte-specific Angpt2 knockout phenotypes. Our findings demonstrate the critical roles of Angpt2-integrin α5β1 signaling in SAT endothelium in regulating whole-body fat distribution for metabolic health and highlight adipocyte-endothelial crosstalk as a potential target for prevention of ectopic lipid deposition-induced lipotoxicity and insulin resistance.

The present study aimed to compare the expression levels of excitatory amino acid transporters (EAATs) and growth status of piglets weaned at 10‑20 days after birth with suckling piglets. A total of 40 hybrid piglets (Landrace x Large White x Duroc) born to 40 different sows, with similar body weight were selected for the present study. They were randomly divided into two groups (n=20 per group): Control group (suckling piglets) and experimental group (weaned piglets, reared in isolation). The experiment lasted for 10 days. At the end of the experiment, 12 piglets were randomly selected from each group and the jejunum and the ileum were collected in order to determine excitatory amino acid carrier 1 (EAAC1) expression levels and free amino acid content. The present study determined that early weaning significantly reduced EAAC1 gene and protein (57 and 73 kDa) expression levels and glutamate transporter associate protein 3‑18 (GTRAP3‑18; 50 kDa) in the jejunum and the ileum compared with the suckling group (P<0.05). Weaning led to an increased content of free glutamic acid (Glu) and total amino acids in the jejunum; however, content of free Glu and total amino acids in the ileum was significantly reduced (P<0.05). Early weaning reduced the expression of EAAC1 and GTRAP3‑18, which was possibly due to the amino acid absorption and transport disorder in the small intestine due to the Glu deficiency.

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.

Amino acid transporters (AATs) are essential integral membrane proteins that serve multiple roles, such as facilitating the transport of amino acids across cell membranes. They play a crucial role in the growth and development of plants. Phaseolus vulgaris, a significant legume crop, serves as a valuable model for studying root symbiosis. In this study, we have conducted an exploration of the AAT gene family in P. vulgaris. In this research, we identified 84 AAT genes within the P. vulgaris genome sequence and categorized them into 12 subfamilies based on their similarity and phylogenetic relationships with AATs found in Arabidopsis and rice. Interestingly, these AAT genes were not evenly distributed across the chromosomes of P. vulgaris . Instead, there was an unusual concentration of these genes located toward the outer edges of chromosomal arms. Upon conducting motif analysis and gene structural analysis, we observed a consistent presence of similar motifs and an intron-exon distribution pattern among the subfamilies. When we analyzed the expression profiles of PvAAT genes, we noted tissue-specific expression patterns. Furthermore, our investigation into AAT gene expression under rhizobial and mycorrhizal symbiotic conditions revealed that certain genes exhibited high levels of expression. Specifically, ATLa5 and LHT2 was notably upregulated under both symbiotic conditions. These findings point towards a potential role of AATs in the context of rhizobial and mycorrhizal symbiosis in P. vulgaris, in addition to their well-established regulatory functions.

Regulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high-level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC-XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3-. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl-/HCO3- exchanger with a suggested role in pancreatic HCO3- secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl-/HCO3- exchange in cells coexpressing SLC26A6 and AT1a-AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption.

Streptococcus iniae (S. iniae) is a major pathogen that causes considerable morbidity and mortality in cultured fish worldwide. The pathogen's ability to adapt to the host affects the extent of infection, hence understanding the mechanisms by which S. iniae overcomes physiological stresses during infection will help to identify potential virulence determinants of streptococcal infection. Grow S. iniae under iron-restricted conditions is one approach for identifying host-specific protein expression. Iron plays an important role in many biological processes but it has low solubility under physiological condition. Many microorganisms have been shown to be able to circumvent this nutritional limitation by forming direct contacts with iron-containing proteins through ATP-binding cassette (ABC) transporters. The ABC transporter superfamilies constitute many different systems that are widespread among living organisms with different functions, such as ligands translocation, mRNA translation, and DNA repair.

The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.

Bacterial capsular polysaccharides are virulence factors and are considered T cell-independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+) T cells in a major histocompatibility complex (MHC) class II-dependent manner. The mechanism of carbohydrate presentation to CD4(+) T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell-dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide-carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.

Measurements of transepithelial potential and resistance in tissue and organ model systems enable the evaluation of the Ni2+ effect on the epithelial sodium channels, aquaporin 3, and the sodium-potassium pump in the epithelial cells. The aim of the presented study was to assess the immediate and prolonged effect of nickel ions on the transport of sodium ions in tissues exposed to direct contact with nickel, including airways, digestive tract and the skin. The influence of 0.1 mM nickel solution was performed on the trachea (n = 34), intestine (n = 44), and skin (n = 51) samples descended from 16 New Zealand albino rabbits. The electrophysiological parameters were measured in a modified Ussing chamber in stationary conditions and during a 15-s mechanical-chemical stimulation. A statistically significant decrease in the electric resistance values and the smallest range of the measured potential were observed for the Ni-treated trachea specimens. The use of nickel solution did not affect the sodium transport in the intestine epithelium. The skin fragments showed altered sodium ion transport, as demonstrated by the lower range and intensity of the measured potential. The gastrointestinal tract seems to be an organ best adapted to contact with nickel ions. In airways, nickel ions most likely enter epithelial cells and the space between them, modifying proteins and the airway surface liquid. The skin turned out to be the most sensitive tissue to the intensification of sodium ion transport through nickel ions.