Mutations in Neurospora crassa which affect multiple amino acid transport systems

Characterization of a double mutant, his-6: hgu-4, which is unable to utilize l-histidyl-glycine as a source of histidine has revealed a new locus on linkage group V. The hgu-4 genotype results in a generalized reduced transport activity for amino acids, with a concomitant increased resistance to amino acid analogs. Transport rates and analog resistance for amino acids by this mutant are compared to the previously reported transport deficient mutants fpr-1, nap and un-3.Transport of l-aspartate as a function of temperature is examined in a variety of transport deficient strains in an attempt to explain the mode of action of mutation which pleiotropically affect several genetically and biochemically distinct amino acid transport systems.     

Taxonomic evaluation of amino acid metabolism inLegionella

Legionella utilizes amino acids as the sole source of carbon and energy. We have examined 62 strains of nine species to determine whether their amino acid metabolism correlated with species definition. Neither definitive species differences nor serogroup differences within the speciesLegionella pneumophila were found. Greatest metabolic activity of all strains was observed with glutamate, alanine, aspartate, and proline; anaerobically, the greatest reduction of methylene blue was obtained with glutamate, aspartate, histidine, and lysine. Within the genus, two major groups were observed: those species that showed little endogenous activity and metabolized a select group of amino acids, and those species that showed strong endogenous activity and appeared to actively metabolize virtually all the amino acids.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

Mutations in Neurospora crassa which affect multiple amino acid transport systems.

Characterization of a double mutant, his-6: hgu-4, which is unable to utilize L-histidyl-glycine as a source of histidine has revealed a new locus on linkage group V. The hgu-4 genotype results in a generalized reduced transport activity for amino acids, with a concomitant increased resistance to amino acid analogs. Transport rates and analog resistance for amino acids by this mutant are compared to the previously reported transport deficient mutants fpr-1, nap and un-3. Transport of L-aspartate as a function of temperature is examined in a variety of transport deficient strains in an attempt to explain the mode of action of mutation which pleiotropically affect several genetically and biochemically distinct amino acid transport systems.     

Growth and amino acid contents of<Emphasis Type="Italic">Spirulina platensis</Emphasis> with different nitrogen sources

The growth and amino acid contents of the cyanobacterium,Spirulina platensis strain NIES 46, were investigated using ammonium, nitrate, nitrite, or urea as the sole nitrogen source in a batch culture. Chlorophylla concentration was highest at 2,096 μg/L in the nitrate group after 10 days of cultivation, while the dry weight ofS. platensis was highest at 4.5 g/L in the ammonium group after 30 days of cultivation. The total amino acid content was highest at 174 mg/g dry weight ofS. platensis in the urea group at the end of the cultivation period, yet the amino acid patterns forS. platensis were similar for all the experimental groups. Therefore, it seemed that the growth and amino acid composition ofS. platensis varied depending on the type of nitrogen sources, while the amino acid patterns were not changed. Also, the most efficient harvesting time forS. platensis seemed to be approximately 10 days after cultivation.     

Mutations in Saccharomyces cerevisiae which confer resistance to several amino acid analogs.

Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline. All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake. In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1. Both raa4 and gap1 prevent uptake of [3H]leucine in liquid cultures. The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.     

Overexpression of GmAAP6a enhances tolerance to low nitrogen and improves seed nitrogen status by optimizing amino acid partitioning in soybean

Amino acid transport via phloem is one of the major source‐to‐sink nitrogen translocation pathways in most plant species. Amino acid permeases (AAPs) play essential roles in amino acid transport between plant cells and subsequent phloem or seed loading. In this study, a soybean AAP gene, annotated as GmAAP6a, was cloned and demonstrated to be significantly induced by nitrogen starvation. Histochemical staining of GmAAP6a:GmAAP6a‐GUS transgenic soybean revealed that GmAAP6a is predominantly expressed in phloem and xylem parenchyma cells. Growth and transport studies using toxic amino acid analogs or single amino acids as a sole nitrogen source suggest that GmAAP6a can selectively absorb and transport neutral and acidic amino acids. Overexpression of GmAAP6a in Arabidopsis and soybean resulted in elevated tolerance to nitrogen limitation. Furthermore, the source‐to‐sink transfer of amino acids in the transgenic soybean was markedly improved under low nitrogen conditions. At the vegetative stage, GmAAP6a‐overexpressing soybean showed significantly increased nitrogen export from source cotyledons and simultaneously enhanced nitrogen import into sink primary leaves. At the reproductive stage, nitrogen import into seeds was greatly enhanced under both sufficient and limited nitrogen conditions. Collectively, our results imply that overexpression of GmAAP6a enhances nitrogen stress tolerance and source‐to‐sink transport and improves seed quality in soybean. Co‐expression of GmAAP6a with genes specialized in source nitrogen recycling and seed loading may represent an interesting application potential in breeding.     

Regulation of pteridine biosynthesis and aromatic amino acid hydroxylation inDrosophila melanogaster

The relationship between high dietary levels of aromatic amino acid and regulation of pteridines inDrosophila eyes was examined by measuring changes in pool levels of six pterins in the wild type and mutants and amino acid pool levels in flies that carry mutations for pteridine biosynthesis. The effect upon relative viability and developmental times was also analyzed; relative viability was affected byl-phenylalanine,l-tryptophan, andl-tyrosine in decreasing order and thed-amino acids had little or no effect. The changes in concentration of biopterin, dihydrobiopterin, pterin, sepiapterin, drosopterins, and isoxanthopterin showed a characteristic pattern of increased and/or decreased amounts in response to each of the threel-amino acids. Pterin was regularly increased, and isoxanthopterin decreased.l-Tyrosine caused a 2.1-fold increase in dihydrobiopterin, the largest increase found in this study;l-tryptophan also caused dihydrobiopterin to increase butl-phenylalanine did not. Of 18 eye-color mutants examined, 2 were found to contain high levels of phenylalanine and/or tyrosine,Pu ² andHn ^r3. These two mutants, along withpr ^c4 cn/pr ^m2b cn, were shown to be very sensitive to dietaryl-phenylalanine, indicating that having low levels of certain pteridines makes them susceptible to toxic effects of these amino acids. Therefore, high levels of aromatic amino acids can perturb the balance among pteridine pools, and low levels of some pteridines in mutants are correlated with the inability to withstand the toxic effects of phenylalanine. From the patterns of change in the pteridines we suggest that tetrahydropterin may also be a cofactor for hydroxylation of phenylalanine, along with tetrahydrobiopterin.This work was sponsored in part by a grant from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae

Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.     

Evolutionary changes reflected by the cellular amino acid composition

Summary Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli,Klebsiella,Proteus, andVibrio alginolyticus) was identical. However, the amino acid composition ofStaphylococcus aureus andBacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium,Chroococidiopsis) was quite similar to that ofE. coli. A marked difference in the amino acid composition was observed betweenE. coli and green alga (Chlorella), and significant differences were observed betweenE. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.     

Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll

Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.     

Transport and utilizing of the biosynthetic intermediate shikimic acid in Escherichia coli

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poort growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced.Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport.Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% contransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not contransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that bot classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (K_m) for shikimate transport.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Breeding and characterization of amino acid-analogue-resistant mutants of Arthrospira platensis

To obtain amino acid-analogue-resistant mutants the wild strain A9 of Arthrospira platensis was mutated by ethylmethane sulfonate (EMS). Mutagenic effects of strain A9 by EMS were studied. The experimental results indicated that the survival rate curve of strain A9 took a typical “exponential shape” with lethal dosage of EMS being 1 %. The survival of A9 strain was 13.2 % when treated with 0.4 % of EMS, and the resistant mutation rates to two amino acid analogues, ρ-fluorophenylalanine (FPA) and L-canavanine sulphate (CS), were greatly increased with the highest rates being at 4.9 × 10^?4 and 3.24 × 10^?4, respectively. By repeated screening, two stable mutants resistant to amino acid analogues, A9f resistant to FPA and A9c resistant to CS, were obtained. Resistances of the two mutants to corresponding amino acid-analogues were both significantly increased. Compared with their parent strain A9, A9f appeared larger than A9 performance in filament diameter, spiral diameter, spiral pitch, filament length and spiral number, and A9c showed much longer length and spiral pitch than those of the initial strain. Analysis results on amino acids compositions and contents showed that both two mutants accumulated quite higher concentration of amino acids in cells. The two mutants might be excellent high amino acids producing strain. By this means two useful mutants with stable genetic makers for further genetic study of A. platensis were obtained, which laid a good foundation for further study on the transformation of A. platensis.     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Activation of a new proline transport system in Salmonella typhimurium.

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.     

The amino acid composition of mammalian and bacterial cells

Summary High performance liquid chromatography was used to analyze the amino acid composition of cells. A total of 17 amino acids was analyzed. This method was used to compare the amino acid compositions of the following combinations: primary culture and established cells, normal and transformed cells, mammalian and bacterial cells, andEscherichia coli andStaphylococcus aureus. The amino acid compositions of mammalian cells were similar, but the amino acid compositions ofEscherichia coli andStaphylococcus aureus differed not only from mammalian cells, but also from each other. It was concluded that amino acid composition is almost independent of cell establishment and cell transformation, and that the amino acid compositions of mammalian and bacterial cells differ. Thus, it is likely that changes in amino acid composition due to cell transformation or species differences between mammalian cells are negligible compared with the differences between mammalian and bacterial cells, which are more distantly related.     

Application of the profiles of amino acid utilization as the sole carbon and nitrogen sources for pseudomonad taxonomy

Profiles of utilization of 20 amino acids were determined for 218 strains of 10 Pseudomonas species (P. aeruginosa, P. putida, P. fluorescens, P. stutzeri, P. alcaligenes, P. pseudoalcaligenes, P. luteola, P. oryzihabitans, P. mendocina, and P. chlororaphis), including the type strains of these species. Amino acid utilization was determined on minimal salt agar with an amino acid as the sole source of nitrogen and carbon. All the investigated pseudomonad species had species-specific profiles of amino acid utilization. For the type strains of all species, Jaccard’s coefficients of community were different (Sj = 0.31–0.82), in accordance with the interspecies differences. The similarity between the intraspecies variants of the profiles and that of the type strain was high; for 98% of P. aeruginosa strains, Sj = 0.85−1.0; for 100% of P. putida, P. stutzeri, and P. alcaligenes strains, Sj was 0.87–1.0, 0.90, and 0.86–1.0, respectively. Only for P. fluorescens and P. pseudoalcaligenes were low Sj of the intraspecies profiles revealed, in accordance with the known phenotypic heterogeneity of these species. These results agree with the known pseudomonad classification, and the method is therefore valid for identification of known species and for determination of the new members of the genus Pseudomonas.