Putative Amino Acid Transporter Research Articles

Salt tolerance of a Sinorhizobium meliloti strain isolated from dry lands: growth capacity and protein profile changes

Sinorhizobium meliloti strain A5, isolated from root nodules of Medicago truncatula growing spontaneously in Tunisian dry lands, showed better growth performance under different sodium chloride concentrations than the standard reference strain RCR2011. Strain A5 has the ability to grow even at 684 mM NaCl. Two-dimensional (2D) electrophoretic analysis of the salt effect on proteome profiles revealed the greatest change on the 4th day of exposure. The halotolerant strain A5 showed more upregulated protein spots than the standard RCR2011 strain (23% of the analyzed protein spots vs only 10% in strain RCR2011) and fewer downregulated protein spots (28% vs 69%). Proteins over-expressed in salty medium as determined by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for both strains were identified as an outer-membrane protein, an l-amino-acid binding periplasmic transporter protein, a 50S ribosomal protein (L25), a heat shock protein, and a putative oxido-reductase protein. Down-regulated proteins were identified as a putative iron-binding periplasmic transporter protein, a putative lipoprotein precursor, a putative spermidine/putrescine-binding periplasmic transporter protein, and a putative amino-acid binding ABC transporter protein. The profile of strain A5 showed higher over-expression of an oxidoreductase and a heat-shock protein, and specifically an induction of a putative oligopeptide ABC transporter ATP-binding protein.

Origin of the Putrescine-Producing Ability of the Coagulase-Negative Bacterium Staphylococcus epidermidis 2015B

A multiplex PCR method, aimed at the detection of genes associated with biogenic amine production, identified the odc gene encoding ornithine decarboxylase in 1 of 15 strains of Staphylococcus epidermidis. The ability of the positive strain, S. epidermidis 2015B, to produce putrescine in vitro was demonstrated by high-performance liquid chromatography (HPLC). In this strain, the odc gene was detected on plasmid DNA, suggesting that the ability to form putrescine is carried by a mobile element, which explains the fact that the trait is strain dependent within the S. epidermidis species. A 6,292-bp nucleotide sequence harboring the putative odc gene was determined. S. epidermidis ornithine decarboxylase (ODC) showed 60 to 65% sequence identity with known ODCs of Gram-positive as well as Gram-negative bacteria. Downstream of the odc gene, a gene encoding a putative amino acid transporter was found that shared 59% sequence identity with the ornithine/putrescine exchanger (PotE) of Escherichia coli. Cloning and expression of the potE gene of S. epidermis 2015B in Lactococcus lactis demonstrated that the gene product transported ornithine and putrescine into the cells and efficiently exchanged putrescine for ornithine. Analysis of the flanking regions showed high identity levels with different S. epidermidis plasmid sequences, which would confirm the plasmidic location of the odc operon. It follows that the odc and potE gene pair encodes a putrescine-producing pathway in S. epidermis 2015B that was acquired through horizontal gene transfer.

Cloning and characterization of the densoviruses susceptible gene + nsd-2 in the silkworm, Bombyx mori

Recently, a silkworm gene responsible for the susceptibility to BmDNV-2 (a parvo-like virus) has been discovered and designated as +nsd-2, which encodes a 12-pass transmembrane protein. BmDNV-Z, isolated in Zhenjiang of China, has a high homology with BmDNV-2 in serological characteristics and genome structure. However, it is still uncertain whether +nsd-2 is also responsible for susceptibility to BmDNV-Z. In this study, we cloned +nsd-2 gene from Chinese silkworm strain (HuaBa35) that is susceptible to BmDNV-Z. DNA sequencing confirmed that +nsd-2 from HuaBa35 is the same with that from NO.908 susceptible to BmDNV-2. RT-PCR analysis showed that the gene +nsd-2 was only expressed in larval midgut and widely expressed in every instar of larva. Bioinformatic study showed that +nsd-2 is a putative amino acid transporter with three glycosylation sites and located on chromosome 11. In addition, +nsd-2 gene was also expressed by baculovirus expression system in Sf9 cell. Our analysis will contribute to the detailed investigation on the infection mechanism of BmDNV.

H+-Independent Glutamine Transport in Plant Root Tips

BackgroundGlutamine is one of the primary amino acids in nitrogen assimilation and often the most abundant amino acid in plant roots. To monitor this important metabolite, a novel genetically encoded fluorescent FRET-reporter was constructed and expressed in Arabidopsis thaliana. As a candidate for the glutamine fluxes, the root tip localized, putative amino acid transporter CAT8 was analyzed and heterologously expressed in yeast and oocytes.Principal FindingsRapid and reversible in vivo fluorescence changes were observed in reporter-expressing root tips upon exposure and removal of glutamine. FRET changes were detected at acid and neutral pH and in the presence of a protonophore, suggesting that part of the glutamine fluxes were independent of the pH. The putative amino acid transporter CAT8 transported glutamine, had a half maximal activity at ∼100 µM and the transport was independent of external pH. CAT8 localized not only to the plasma membrane, but additionally to the tonoplast, when tagged with GFP. Ultrastructural analysis confirmed this dual localization and additionally identified CAT8 in membranes of autophagosomes. Loss-of function of CAT8 did not affect growth in various conditions, but over-expressor plants had increased sensitivity to a structural substrate analog, the glutamine synthetase inhibitor L-methionine sulfoximine.ConclusionsThe combined data suggest that proton-independent glutamine facilitators exist in root tips.

A putative amino acid transporter of the solute carrier 6 family is upregulated by lithium and is required for resistance to lithium toxicity in Drosophila

Lithium is an efficacious drug for the treatment of mood disorders, and its application is also considered a potential therapy for brain damage. However, the mechanisms underlying lithium's therapeutic action and toxic effects in the nervous system remain largely elusive. Here we report on the use of a versatile genetic model, the fruit fly Drosophila melanogaster, to discover novel molecular components involved in the lithium-responsive neurobiological process. We previously identified CG15088, which encodes a putative nutrient amino acid transporter of the solute carrier 6 (SLC6) family, as one of the genes most significantly upregulated in response to lithium treatment. This gene was the only SLC6 gene induced by lithium, and was thus designated as Lithium-inducible SLC6 transporter or List. Either RNA interference (RNAi)–mediated knockdown or complete deletion of List resulted in a remarkable increase in the susceptibility of adult flies to lithium's toxic effects, whereas transgenic expression of wild-type List significantly suppressed the lithium hypersensitive phenotype of List-deficient flies. Other ions such as sodium, potassium and chloride did not induce List upregulation, nor did they affect the viability of flies with suppressed List expression. These results indicate that lithium's biochemical or physical properties, rather than general osmotic responses, are responsible for the lithium-induced upregulation of List, as well as for the lithium-susceptible phenotype observed in List knockdown flies. Interestingly, flies became significantly more susceptible to lithium toxicity when List RNAi was specifically expressed in glia than when it was expressed in neurons or muscles, which is consistent with potential glial expression of List. These results show that the List transporter confers resistance to lithium toxicity, possibly as a consequence of its amino acid transporter activity in CNS glia. Our results have provided a new avenue of investigation toward a better understanding of the molecular and cellular mechanisms that underlie lithium-responsive neurobiological process.

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized l-histidine, l-glutamine, l-proline, l-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Effects of lithium chloride on the gene expression profiles in Drosophila heads

To gain insight into the basic neurobiological processes regulated by lithium—an effective drug for bipolar disorder—we used Affymetrix Genome Arrays to examine lithium-induced changes in genome-wide gene expression profiles of head mRNA from the genetic model organism Drosophila melanogaster. First, to identify the individual genes whose transcript levels are most significantly altered by lithium, we analyzed the microarray data with stringent criteria (fold change > 2; p < 0.001) and evaluated the results by RT-PCR. This analysis identified 12 genes that encode proteins with various biological functions, including an enzyme responsible for amino acid metabolism and a putative amino acid transporter. Second, to uncover the biological pathways involved in lithium's action in the nervous system, we used less stringent criteria (fold change > 1.2; FDR < 0.05) and assigned the identified 66 lithium-responsive genes to biological pathways using DAVID (Database for Annotation, Visualization and Integrated Discovery). The gene ontology categories most significantly affected by lithium were amino acid metabolic processes. Taken together, these data suggest that amino acid metabolism is important for lithium's actions in the nervous system, and lay a foundation for future functional studies of lithium-responsive neurobiological processes using the versatile molecular and genetic tools that are available in Drosophila.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of ~60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

Characterisation of a developmentally regulated amino acid transporter gene from Leishmania amazonensis

The metabolism of protozoan parasites of the Leishmania genus is strongly based on amino acid consumption, but little is known about amino acid uptake in these organisms. In the present work, we identified a Leishmania amazonensis gene (La-PAT1) encoding a putative amino acid transporter that belongs to the amino acid/auxin permease family, a group of H +/amino acid symporters. This single copy gene is upregulated in amastigotes, the life cycle stage found in the mammalian host. La-PAT1 putative orthologous sequences were identified in Leishmania infantum, Leishmania donovani, Leishmania major and Trypanosoma.

Post genomic analysis of permeases from the amino acid/auxin family in protozoan parasites

The “amino acid/auxin permeases” is probably the most represented family of transporters in the Trypanosoma cruzi genome. Using a high-throughput searching routine and preliminary data from the T. cruzi genome project, more than 15,000 sequences were iteratively assembled into contigs, and 60 open reading frames corresponding to different putative amino acid transporters, clustered in 12 groups, were detected and characterized in silico. T. cruzi genomic organization of such sequences showed that these putative amino acid transporter genes are in an unusually large number and arranged in repeat clusters comprising about 0.2% of the genome. These data suggest that the family has evolved following tandem duplication events and constitutes a novel family of variable proteins in protozoan organisms. The mRNA expression of the predicted genes was demonstrated in infective and non-infective parasite forms. Orthologous sequences were also identified in other unicellular parasites such as Leishmania spp., Plasmodium spp., and Trypanosoma brucei.

Do cluster roots of Hakea actities (Proteaceae) acquire complex organic nitrogen?

Although there has been speculation that cluster roots play a role in plant nitrogen (N) acquisition there have been few experimental studies, demonstrating this. We grew the subtropical wet heathland plant Hakea actities in sand and in axenic vermiculite-sand cultures to investigate its use of different N sources. Seedlings produced greater quantities of cluster roots when grown with NO3−, glutathione or protein as N sources than with NH4+, glutamine or without N addition, suggesting that N source influences cluster root initiation and development. Axenically grown seedlings acquired more N when grown with 4.5 mM than with 0.5 mM glutathione or protein as N sources suggesting that seedlings are able to assimilate complex organic N. However, control seedlings that did not receive N other than seed storage N (approximately 0.2 mg N) acquired 2 mg N from an unknown source, so that data are not unequivocal. Peptidase activity in extracts derived from non-axenic cluster root tissue changed with age of cluster roots. Developing and mature cluster roots had higher peptidase activity than young and senescing cluster roots. To determine whether peptidases are associated with the outer surface of cluster roots, entire cluster roots were gently centrifuged and the resulting solution analysed for peptidase activity using gelatine-containing PAGE. Different peptidases were active at different times of cluster root development, suggesting that plant or microbially derived peptidases could play a role in organic N acquisition by cluster roots. Roots and cluster roots expressed a putative amino acid transporter (HaAAT4-1) and peptide transporter (HaPepT1) as determined by northern blot analysis. Expression of HaPepT1 in cluster roots increased throughout cluster root development although further studies need to confirm this.

Identification of a Pathway for the Utilization of the Amadori Product Fructoselysine in Escherichia coli

Escherichia coli was found to grow on fructoselysine as an energetic substrate at a rate of about one-third of that observed with glucose. Extracts of cells grown on fructoselysine catalyzed in the presence of ATP the phosphorylation of fructoselysine and a delayed formation of glucose 6-phosphate from this substrate. Data base searches allowed us to identify an operon containing a putative kinase (YhfQ) belonging to the PfkB/ ribokinase family, a putative deglycase (YhfN), homologous to the isomerase domain of glucosamine-6-phosphate synthase, and a putative cationic amino acid transporter (YhfM). The proteins encoded by YhfQ and YhfN were overexpressed in E. coli, purified, and shown to catalyze the ATP-dependent phosphorylation of fructoselysine to a product identified as fructoselysine 6-phosphate by 31P NMR (YhfQ), and the reversible conversion of fructoselysine 6-phosphate and water to lysine and glucose 6-phosphate (YhfN). The K(m) of the kinase for fructoselysine amounted to 18 microm, and the K(m) of the deglycase for fructoselysine 6-phosphate, to 0.4 mm. A value of 0.15 m was found for the equilibrium constant of the deglycase reaction. The kinase and the deglycase were both induced when E. coli was grown on fructoselysine and then reached activities sufficient to account for the rate of fructoselysine utilization.

Minidiscs encodes a putative amino acid transporter subunit required non-autonomously for imaginal cell proliferation

Drosophila minidiscs mutant larvae have smaller imaginal discs than wild-type larvae. However, transplantation experiments have revealed that minidiscs mutant imaginal discs can grow if cultured in non-mutant hosts. These data suggest that minidiscs is required in one or more non-imaginal tissues for synthesis and/or secretion of a diffusible factor that stimulates imaginal cell proliferation. The 2.3 kb minidiscs transcript accumulates in the larval fat body and encodes a protein containing 12 putative membrane spanning domains that is similar in sequence to amino acid transporter subunits from other eukaryotes, including humans. We propose that in response to amino acid uptake by the transporter encoded by minidiscs, the fat body secretes a diffusible factor required for imaginal disc proliferation.

Amino acid carriers of Ricinus communis expressed during seedling development: molecular cloning and expression analysis of two putative amino acid transporters, RcAAP1 and RcAAP2.

This study reports on the isolation of two putative amino acid carrier cDNAs, RcAAP1 and RcAAP2, from Ricinus communis. Northern analysis shows that RcAAP1 and RcAAP2 are expressed abundantly in the cotyledon and root tissues of developing seedlings and at lower levels in the endosperm and hypocotyl. In the mature plant low expression was observed in the source and sink leaves. We have further characterized the expression of RcAAP1 in Ricinus roots by in situ hybridization. The transcripts are localized in many cell types of the root tip region, including the epidermal and cortical cells, but the highest expression was observed in the cells of the stele situated adjacent to the xylem poles. This is the first report describing the cellular expression of an amino acid transporter in roots, and the results are discussed in relation to the physiological role of this transporter.

A Putative Amino Acid Transporter Is Specifically Expressed in Haustoria of the Rust Fungus Uromyces fabae

A cDNA library constructed from haustoria of the rust fungus Uromyces fabae was screened for clones that are differentially expressed in haustoria. One family of cDNAs (in planta-induced gene 2 [PIG2] was isolated and found to encode a protein with high homologies to fungal amino acid transporters. A cDNA clone containing the complete coding region of PIG2 and the corresponding genomic clone were isolated and sequenced, revealing the presence of 17 introns in the PIG2 gene. Expression of PIG2 mRNA appeared to be restricted to haustoria. With antibodies raised against synthetic peptides, the PIG2-encoded protein was found in membranes fractions of isolated haustoria but not of germinated rust spores. With immunofluorescence microscopy, the putative amino acid transporter was localized to plasma membranes of the haustorial bodies, but not detected in the haustorial neck, haustorial mother cells, or intercellular fungal hyphae growing within infected leaf tissue. These data present for the first time molecular evidence that the rust haustorium plays a special role in the uptake of nutrients from an infected host cell.

Structure and specific expression of a Nicotiana sylvestris putative amino-acid transporter gene in mature and in vitro germinating pollen.

We report here the structure and expression analysis of a Nicotiana sylvestris gene, called nsaap1, coding for a protein which shows significant identity to the members of the Arabidopsis thaliana amino acid permease family. The nsaap1 gene is expressed exclusively in binucleate pollen. Its transcripts begin to accumulate after first pollen mitosis and dramatically increase in mature pollen shortly before anthesis and during in vitro pollen germination. The nsaap1 5'-flanking region contains long regions homologous to the promoter region of the tobacco pollen specific eIF-4A8 translation factor. Our results support the occurrence of an amino acid remobilization in binucleate pollen. The nsaap1 gene could play a role in exchanges between the vegetative and the generative pollen cells.

P2-264 - Molecular cloning and characterization of a putative cationic amino acid transporter from rat brain

P2-264 - Molecular cloning and characterization of a putative cationic amino acid transporter from rat brain

P-633 - Molecular cloning of a putative cationic amino acid transporter from rat brain.

P-633 - Molecular cloning of a putative cationic amino acid transporter from rat brain.