Gas channels for NH(3): proteins from hyperthermophiles complement an Escherichia coli mutant

Ammonium transport (Amt) proteins appear to be bidirectional channels for NH(3). The amt genes of the hyperthermophiles Aquifex aeolicus and Methanococcus jannaschii complement enteric amtB mutants for growth at 25 nM NH(3) at 37 degrees C. To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium. Despite low expression levels, His-tagged Aquifex Amt could be purified by heating and nickel chelate affinity chromatography. It could be studied genetically in Escherichia coli.     

The AMT1 arginine methyltransferase gene is important for plant infection and normal hyphal growth in Fusarium graminearum

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.     

Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and complementation by the cloned gene.

During nitrogen-limited growth, Escherichia coli expresses a specific ammonium or methylammonium ion transport system (Amt). Strains carrying defects in Amt have been isolated following Tn10 transposon mutagenesis. These mutants have less than 10% of the transport activity of the parental strain. Glutamate, glutamine, arginine, or high levels (20 mM) of ammonium will serve as the sole nitrogen source for growth of these strains, and glutamine synthetase is normally expressed and repressed by the nitrogen regulatory (Ntr) system. When transformed with plasmid pGln84, containing lacZ fused to an Ntr promoter (glnLp), the Amt mutants expressed a normal level of beta-galactosidase. Furthermore, P1 bacteriophage transduction of the amt mutation into an Ntr mutant, normally constitutive for Amt, gave Amt- transductants. Therefore, the mutations are unlikely to lie within genes affecting Ntr elements. Following transformation with plasmid libraries of E. coli genomic DNA constructed in pUC9, two plasmids conferring the Amt+ phenotype on the amt mutants were isolated. These plasmids were unable to complement the Amt- phenotype of Ntr- mutants. Restriction digestion of these plasmids revealed common fragments, and Southern blot analyses indicated that the Amt-complementing sequence and the site of Tn10 insertion in the genome occur in the same 3.4-kilobase HindIII-SalI fragment. Insertion of TnphoA into this fragment produced amt::phoA fusions which gave high levels of alkaline phosphatase under nitrogen-limiting conditions but low levels during ammonia excess. This suggests that the amt product contains domains which are exported to the periplasm.     

Role of the Escherichia coli glnALG operon in regulation of ammonium transport.

Escherichia coli expresses a specific ammonium (methylammonium) transport system (Amt) when cultured with glutamate or glutamine as the nitrogen source. Over 95% of this Amt activity is repressed by growth of wild-type cells on media containing ammonia. The control of Amt expression was studied with strains containing specific mutations in the glnALG operon. GlnA- (glutamine synthetase deficient) mutants, which contain polar mutations on glnL and glnG genes and therefore have the Reg- phenotype (fail to turn on nitrogen-regulated operons such as histidase), expressed less than 10% of the Amt activity observed for the parental strain. Similarly, low levels of Amt were found in GlnG mutants having the GlnA+ Reg- phenotype. However, GlnA- RegC mutants (a phenotype constitutive for histidase) contained over 70% of the parental Amt activity. At steady-state levels, GlnA- RegC mutants accumulated chemically unaltered [14C]methylammonium against a 60- to 80-fold concentration gradient, whereas the labeled substrate was trapped within parental cells as gamma-glutamylmethylamide. GlnL Reg- mutants (normal glutamine synthetase regulation) had less than 4% of the Amt activity observed for the parental strain. However, the Amt activity of GlnL RegC mutants was slightly higher than that of the parental strain and was not repressed during growth of cells in media containing ammonia. These findings demonstrate that glutamine synthetase is not required for Amt in E. coli. The loss of Amt in certain GlnA- strains is due to polar effects on glnL and glnG genes, whose products are involved in expression of nitrogen-regulated genes, including that for Amt.     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

Molecular characterization,function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS,NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum

External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.     

Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots

In Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of (15)N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wild-type plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.     

Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH(3) across the cytoplasmic membrane

Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)). Transport and accumulation of the ammonium analog [(14)C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH(4)(+). In bacteria, however, ATP-dependent conversion of methylammonium to gamma-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A Deltavph1 mutant of S. cerevisiae and a Deltavma1 mutant, which lack vacuolar H(+)-ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH(3)NH(3)(+) in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH(3)NH(2). By inference, their natural substrate is NH(3), a gas. We postulate that MEP proteins facilitate diffusion of NH(3) across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO(2).     

Functional analysis of an Arabidopsis T-DNA "knockout" of the high-affinity NH4(+) transporter AtAMT1;1

NH(4)(+) acquisition by plant roots is thought to involve members of the NH(4)(+) transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH(4)(+). Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH(4)(+) influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity (13)NH(4)(+) influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity (13)NH(4)(+) influx in the mutant by 30% compared with wild-type plants, whereas low-affinity (13)NH(4)(+) influx (250 microM-10 mM NH(4)(+)) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH(4)(+) and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH(4)(+) transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.     

Ammonium (methylammonium) transport by heterocysts and vegetative cells of Anabaena variabilis

Transport of the ammonium analogue [(14)C]methylammonium was similar in non-growing, fully differentiated heterocysts as compared to vegetative, multiplying cells of the filamentous cyanobacterium Anabaena variabilis. NH(4)(+) inhibited uptake into the cells and released accumulated methylammonium from the cells. These observations suggest that the main function of ammonium transport in heterocysts may not be NH(4)(+) acquisition but cyclic retention of ammonia produced by nitrogenase.     

Role of AMT1;1 in NH4+ acquisition in Arabidopsis thaliana

AtAMT1;1 was the founding member of the family of AMT/Rh ammonium transporters and accounts for about one third of the total ammonium absorption in the roots of the model plant Arabidopsis. Recent evidence suggested that at least some AMT/Rh proteins are NH3 gas channels. In order to evaluate the transported form of ammonium in AtAMT1;1, the protein was functionally expressed in Xenopus oocytes. AtAMT1;1 elicited NH4+ and methylammonium (MeA+) inward currents that saturated in a voltage-dependent manner with a half maximal concentration of 2.7 +/- 1.6 microM for NH4+ and 5.0 +/- 0.7 microM for the transport analogue methylammonium. AtAMT1;1 was plasma membrane localized and expressed in the root cortex and epidermis, including root hairs. The AtAMT1;1-GFP fusion construct under control of its endogenous promoter revealed additional localization of the protein in the pericycle, in the leaf epidermis, and in mesophyll cells. The functional data and its localization suggest that AtAMT1;1 participates in concentrative NH4+ acquisition in roots, in long-distance transport to the shoots, and in re-uptake of apoplastic NH4+ that derives from photorespiration in shoots.     

Recombinants between Clock Mutants of CHLAMYDOMONAS REINHARDI

Mutants affecting the period length of the biological clock in Chlamydomonas reinhardi have been isolated and a start has been made on analyzing the genetics of this system. In four mutants, the long period characteristic seems to be controlled by single genes at separate loci. Crosses between single mutants, as well as crosses involving three or four mutant genes, yielded progeny with periods characteristic of the parents as well as recombinant types, including normal period (wild type) and extra-long periods (double, triple and quadruple mutants). It was found that the period lengthening effect is additive; that is, the period of double mutants is lengthened by the sum of the period lengthening of the single mutants.     

The molecular physiology of ammonium uptake and retrieval

Plants are able to take up ammonium from the soil, or through symbiotic interactions with microorganisms, via the root system. Using functional complementation of yeast mutants, it has been possible to identify a new class of membrane proteins, the ammonium transporter/methylammonium permease (AMT/MEP) family, that mediate secondary active ammonium uptake in eukaryotic and prokaryotic organisms. In plants, the AMT gene family can be subdivided according to their amino-acid sequences into three subfamilies: a large subfamily of AMT1 genes and two additional subfamilies each with single members (LeAMT1;3 from tomato and AtAMT2;1 from Arabidopsis thaliana). These transporters vary especially in their kinetic properties and regulatory mechanism. High-affinity transporters are induced in nitrogen-starved roots, whereas other transporters may be considered as the 'work horses' that are active when conditions are conducive to ammonium assimilation. The expression of several AMTs in root hairs further supports a role in nutrient acquisition. These studies provide basic information that will be needed for the dissection of nitrogen uptake by plants at the molecular level and for determining the role of individual AMTs in nutrient uptake and potentially in nutrient efficiency.     

Natural history of transposition in the green alga Chlamydomonas reinhardtii: use of the AMT4 locus as an experimental system

The AMT4 locus of the green alga Chlamydomonas reinhardtii, which we mapped to the long arm of chromosome 8, provides a good experimental system for the study of transposition. Most mutations that confer resistance to the toxic ammonium analog methylammonium are in AMT4 and a high proportion of spontaneous mutations are caused by transposon-related events. Among the 15 such events that we have characterized at the molecular level, 9 were associated with insertions of the retrotransposon TOC1, 2 with a small Gulliver-related transposon, and 1 with the Tcr1 transposon. We found that Tcr1 is apparently a foldback transposon with terminal inverted repeats that are much longer and more complex than previously realized. A duplication of Tcr1 yielded a configuration thought to be important for chromosomal evolution. Other mutations in AMT4 were caused by two mobile elements that have not been described before. The sequence of one, which we propose to call the Bill element, indicates that it probably transposes by way of a DNA intermediate and requires functions that it does not encode. The sequence of the other and bioinformatic analysis indicates that it derives from a miniature retrotransposon or TRIM, which we propose to call MRC1 (miniature retrotransposon of Chlamydomonas).     

The Amt/Mep/Rh family of ammonium transport proteins

The Amt/Mep/Rh family of integral membrane proteins comprises ammonium transporters of bacteria, archaea and eukarya, as well as the Rhesus proteins found in animals. They play a central role in the uptake of reduced nitrogen for biosynthetic purposes, in energy metabolism, or in renal excretion. Recent structural information on two prokaryotic Amt proteins has significantly contributed to our understanding of this class, but basic questions concerning the transport mechanism and the nature of the transported substrate, NH3 or [NH4(+)], remain to be answered. Here we review functional and structural studies on Amt proteins and discuss the bioenergetic issues raised by the various mechanistic proposals present in the literature.