Expression of Genes Encoding the RhtB Family Proteins Depends on the Global Regulator Lrp

In this work, we continued to study the genes encoding the RhtB family proteins. We studied regulation of four genes of this family: rhtB, rhtC, yeaS, and yahN, two of which (rhtB and rhtC) were previously shown to be involved in amino acid efflux from cells. The results of this study showed that the expression of these genes is regulated by the global regulator Lrp; it depends on the presence of certain amino acids in the growth medium and increases in certain types of physiological stress.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 374–378.Original Russian Text Copyright © 2005 by Kutukova, Zakataeva, Livshits.     

The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression

Overexpression of the yeaS gene encoding a protein belonging to the RhtB transporter family conferred upon cells resistance to glycyl-l-leucine, leucine analogues, several amino acids and their analogues. yeaS overexpression promoted leucine and, to a lesser extent, methionine and histidine accumulation by the respective producing strains. Our results indicate that yeaS encodes an exporter of leucine and some other structurally unrelated amino acids. The expression of yeaS (renamed leuE for "leucine export") was induced by leucine, l-alpha-amino-n-butyric acid and, to a lesser extent, by several other amino acids. The global regulator Lrp mediated this induction.     

Export of metabolites by the proteins of the DMT and RhtB families and its possible role in intercellular communication

The earlier published and new experimental data are summarized on the properties of the genes encoding the membrane proteins of the DMT family (RhtA (YbiF), EamA (YdeD), YijE, YddG, YedA, PecM, eukaryotic nucleoside phosphate sugar and hexose phosphate transporters), the RhtB/LysE family (RhtB, RhtC, LeuE, YahN, EamB (YfiK), ArgO (YggA), CmaU), as well as some other families (YicM, YdhC, YdeAB, YdhE (NorE)). These proteins are involved in the export of amino acids, purines, and other metabolites from the cell. The expression of most of the genes encoding these proteins is not induced by the substrates they transport but is controlled by the global regulation systems, such as the Lrp protein, and activated by the signal compounds involved in the intracellular communication. The level of expression, assessed in experiments on translational fusion of the corresponding bacterial genes with the beta-galactosidase gene, depends on the growth phase of the bacterial culture, composition of the medium, and some stress factors, such as pH osmolarity or decreased aeration. The efflux of normal cell metabolites is assumed to be the natural function of these proteins. This function may play a role in density-dependent behavior of cell populations (quorum sensing). It may have been enhanced in the course of evolution via specialization of these proteins in the efflux of compounds derived from metabolic intermediates and adjusted to the role of transmitters.     

Export of metabolites by the proteins of the DMT and RhtB families and its possible role in intercellular communication

The earlier published and new experimental data are summarized on the properties of the genes encoding the membrane proteins of the DMT family (RhtA (YbiF), EamA (YdeD), YijE, YddG, YedA, PecM, eukaryotic nucleotide sugar, triose phosphate/phosphate, and hexose phosphate transporters), the RhtB/LysE family (RhtB, RhtC, LeuE, YahN, EamB (YfiK), ArgO (YggA), CmaU), as well as some other families (YicM, YdhC, YdeAB, YdhE (NorE)). These proteins are involved in the export of amino acids, purines, and other metabolites from the cell. The expression of most of the genes encoding these proteins is not induced by the substrates they transport but is controlled by the global regulation systems, such as the Lrp protein, and activated by the signal compounds involved in the intracellular communication. The level of expression, assessed in experiments on translational fusion of the corresponding bacterial genes with the β-galactosidase gene, depends on the growth phase of the bacterial culture, composition of the medium, and some stress factors, such as pH, osmolarity or decreased aeration. The efflux of normal cell metabolites is assumed to be the natural function of these proteins. This function may play a role in density-dependent behavior of cell populations (quorum sensing). It may have been enhanced in the course of evolution via specialization of these proteins in the efflux of compounds derived from metabolic intermediates and adjusted to the role of transmitters.     

Influence of threonine exporters on threonine production in Escherichia coli

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.     

New ubiquitous translocators: amino acid export by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.     

Identification of a new expanding family of genes characterized by atypical LRR domains. Localization of a cluster preferentially expressed in oocyte

In the present work, we have used the in silico subtraction methodology to identify novel oocyte-specific genes in the mouse. By this way, we have identified in silico a new family of genes composed of more than 80 members. Sequence analysis showed that these genes belong to the superfamily of leucine-rich repeat (LRR) proteins. However, LRRs of this family display some variability in length and in amino acids composition within the beta-strands region, as more leucine residues are substituted by other hydrophobic amino acids as compared to canonical LRRs. Interestingly, for nine of these genes, the ESTs were represented almost exclusively in mouse egg libraries. Three of them were selected for experimental study. By RT-PCR and in situ hybridization, we confirmed their specific expression in the mouse oocyte from primary to preovulatory follicles. These three genes are localized in a cluster on mouse chromosome 4, in the vicinity of another recently discovered oocyte specific gene called oogenesin, that we also found to belong to the same family. We thus re-named this latter gene 'oogenesin-1', and the three genes identified here were named oogenesin-2, -3 and -4.     

Characterization of a sterile dwarf mutant and the cloning of zeaxanthin epoxidase in Asian cotton (<Emphasis Type="Italic">Gossypium arboreum</Emphasis> L.)

Amino acids are constituents of proteins, precursors of many secondary metabolites and nitrogen carriers in plants. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. However, the amino acid transport in tea plant is rarely reported. In this study, six cationic amino acid transporter (CAT) family genes were cloned. Phylogenetic analysis categorized these CsCATs into four subgroups. These CsCATs all contain the 12–14 transmembrane domains and the conserved CAT motifs. Their expression was tissue-specific, with higher expression levels in root and stem and correlated to the abundances of key free amino acids such as Theanine. Some CsCATs expression responded to some abiotic stress conditions and to the exogenous application of theanine (Thea), glutamine or ethylamine hydrochloride, an ethylamine precursor for Thea biosynthesis. Our results indicated that the CsCATs expression is regulated by amino acid contents and is sensitive to abiotic stresses. These findings shed light on the mechanism of amino acid transport in tea plants.     

Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

Expression profiling of the solute carrier gene family in chicken intestine from the late embryonic to early post-hatch stages

Intestinal development during late embryogenesis and early post-hatch has a long-term influence on digestive and absorptive capacity in chickens. The objective of this research was to obtain a global view of intestinal solute carrier (SLC) gene family member expression from late embryogenesis until 2 weeks post-hatch with a focus on SLC genes involved in uptake of sugars and amino acids. Small intestine samples from male chicks were collected on embryonic days 18 (E18) and 20 (E20), day of hatch and days 1, 3, 7 and 14 post-hatch. The expression profiles of 162 SLC genes belonging to 41 SLC families were determined using Affymetrix chicken genome microarrays. The majority of SLC genes showed little or no difference in level of expression during E18–D14. A number of well-known intestinal transporters were upregulated between E18 and D14 including the amino acid transporters rBAT , y ⁺ LAT-2 and EAAT3 , the peptide transporter PepT1 and the sugar transporters SGLT1 , GLUT2 and GLUT5 . The amino acid transporters CAT-1 and CAT-2 were downregulated. In addition, several glucose and amino acid transporters that are novel to our understanding of nutrient absorption in the chicken intestine were discovered through the arrays ( SGLT6 , SNAT1 , SNAT2 and AST ). These results represent a comprehensive characterization of the expression profiles of the SLC family of genes at different stages of development in the chicken intestine and lay the ground work for future nutritional studies.     

楸树CbuATX1，CbuATX1-like和CbuATX2基因克隆及生物信息学分析

开花是高等植物内部遗传因素和外界环境因素共同调节完成的复杂生命过程,是植物进入生殖生长从而具备遗传能力和繁殖能力的象征。使用PCR技术,以楸树（Catalpa bungei）混合芽cDNA为模板分别克隆CbuATX1,CbuATX1-like和CbuATX2 3个基因,并利用相关生物信息学软件对这3个基因编码的蛋白质结构进行预测,同时对基因的启动子序列进行顺式作用元件分析,通过qRT-PCR技术检测3个基因在楸树混合芽不同发育时期的表达量。结果表明：CbuATX1的CDS全长为726 bp,编码241个氨基酸,存在跨膜运输结构,属于HMA蛋白家族,与芝麻（Sesamum indicum）、甜菜（Beta vulgaris）亲缘关系较近。CbuATX1-like的CDS全长为801 bp,编码266个氨基酸,存在跨膜运输结构,属于HMA蛋白家族,与胡萝卜（Daucus carota）、欧洲橄榄（Olea europaea）亲缘关系较近。CbuATX2的CDS全长为1 554 bp,编码517个氨基酸,不存在跨膜运输结构,属于PWWP蛋白家族,与马尾草（Erythranthe guttatus）亲缘关系较近。这3个基因启动子均含有多个真核生物启动子的基本元件,如CAAT-box和TATA-box等,此外,还含有光响应、低温响应、生长素响应,以及与干旱诱导相关的MYB结合位点等3个基因与光响应密切相关,还可能参与外界环境胁迫响应等过程。qRT-PCR结果显示,上述3个基因在1年生普通楸树无性系9-1和突变株系-百日花楸树不同发育时期的表达量呈现显著差异。通过以上研究以期能够进一步阐述百日花楸树开花的性状,为楸树开花机制的研究和楸树的定向遗传改良提供理论支持。     

Molecular and functional characterization of a family of amino acid transporters from Arabidopsis

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.     

Gdf16, a novel member of the growth/differentiation factor subgroup of the TGF-beta superfamily, is expressed in the hindbrain and epibranchial placodes

We have isolated and characterized the developmental expression of Xenopus gdf16, a novel member of the growth/differentiation factor (gdf) gene family. The gdf16 gene encodes a pre-proprotein of 413 amino acids and a mature peptide of 122 amino acids. Gdf16 is most closely related to the zebrafish genes dynamo and radar, but exhibits a completely different expression pattern. Gene expression is detected at early tailbud (stage 25) in the first two epibranchial placodes and in a hindbrain-specific domain. As development proceeds, the gene is expressed in all the epibranchial placodes, the hindbrain, and the diencephalon.     

Amino acids stimulate glycyl-tRNA synthetase nuclear localization for mammalian target of rapamycin expression in bovine mammary epithelial cells

Amino acids are required for the activation of mammalian target of rapamycin (mTOR) to increase cell growth, protein and lipid synthesis, and inhibit autophagy. However, the mechanism through which amino acids activate the mTOR signaling is still largely unknown. In our previous study, we discovered that glycyl-tRNA synthetase (GlyRS) is a key mediator of amino-acid-induced mTOR expression and activation in bovine mammary epithelial cells (BMECs). Here we show that amino acids stimulate GlyRS nuclear localization for mTOR expression in BMECs. Met stimulates GlyRS nuclear localization, and the nuclear GlyRS is cleaved into a C-terminus-containing truncated form. We prove that GlyRS has a bipartite nuclear leading sequences, and GlyRS is phosphorylated at Thr544 and Ser704 in the cytoplasm under the stimulation of amino acids (Met, Leu, and Lys). The nuclear GlyRS physically binds to nuclear factor kappa B1, triggers its phosphorylation, thereby enhancing mRNA expression of its target genes including mTOR, S6K1, and 4EBP1. We further demonstrate that GlyRS is required for the inhibition of autophagy by Met. Thus our work elucidates that amino acids trigger GlyRS phosphorylation and nuclear localization to enhance the mRNA expression of mTOR.     

The novel transmembrane Escherichia coli proteins involved in the amino acid efflux.

A novel gene of Escherichia coli, rhtB, has been characterized. Amplification of this gene provides resistance to homoserine and homoserine lactone. Another E. coli gene, rhtC, provides resistance to threonine. The homologues of RhtB are widely distributed among various eubacteria and archaea, from one to 12 copies of family members that differ in their primary structure were found in the genomes. Most of them are genes that encode hypothetical transmembrane proteins. Experimental data that indicate participation of the rhtB product in the excretion of homoserine have been obtained.     

A strong effect of AT mutational bias on amino acid usage in Buchnera is mitigated at high-expression genes

The advent of full genome sequences provides exceptionally rich data sets to explore molecular and evolutionary mechanisms that shape divergence among and within genomes. In this study, we use multivariate analysis to determine the processes driving genome-wide patterns of amino usage in the obligate endosymbiont Buchnera and its close free-living relative Escherichia coli. In the AT-rich Buchnera genome, the primary source of variation in amino acid usage differentiates high- and low-expression genes. Amino acids of high-expression Buchnera genes are generally less aromatic and use relatively GC-rich codons, suggesting that selection against aromatic amino acids and against amino acids with AT-rich codons is stronger in high-expression genes. Selection to maintain hydrophobic amino acids in integral membrane proteins is a primary factor driving protein evolution in E. coli but is a secondary factor in Buchnera. In E. coli, gene expression is a secondary force driving amino acid usage, and a correlation with tRNA abundance suggests that translational selection contributes to this effect. Although this and previous studies demonstrate that AT mutational bias and genetic drift influence amino acid usage in Buchnera, this genome-wide analysis argues that selection is sufficient to affect the amino acid content of proteins with different expression and hydropathy levels.