Acid Transport Mutant Research Articles

Differential Interference with Pythium ultimum Sporangial Activation and Germination by Enterobacter cloacae in the Corn and Cucumber Spermospheres

Differential protection of plants by Enterobacter cloacae was studied by investigating early sensing and response behavior of Pythium ultimum sporangia toward seeds in the presence or absence of E. cloacae. Ten percent of P. ultimum sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of E. cloacae (EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of E. cloacae (strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of E. cloacae failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of E. cloacae strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of P. ultimum on cucumber seeds treated with E. cloacae EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds, P. ultimum biomass was not affected by the presence of any E. cloacae strain. When introduced after sporangial activation had occurred, E. cloacae failed to reduce P. ultimum biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of P. ultimum and the number of activated and germinated sporangia in the spermosphere, suggesting that E. cloacae suppresses P. ultimum seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.

ANT1, an aromatic and neutral amino acid transporter in Arabidopsis.

A new amino acid transporter was identified from the Arabidopsis expressed sequence tag cDNAs by expressing the cDNA in a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast showed that it transports aromatic and neutral amino acids, as well as arginine. This transporter (ANT1, aromatic and neutral transporter) also transports indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The cDNA is 1.6 kb in length with an open reading frame that codes for a protein with 432 amino acids and a calculated molecular mass of 50 kD. Hydropathy analysis showed ANT1 is an integral membrane protein with 11 putative membrane-spanning domains. Southern analysis and a BLAST search of the Arabidopsis genome database suggests that ANT1 is part of a small gene family containing at least five members. Phylogenetic comparisons with other known amino acid transporters in plants suggests that ANT1 represents a new class of amino acid transporter. RNA gel-blot analysis showed that this transporter is expressed in all organs with highest abundance in flowers and cauline leaves.

LHT1, a lysine- and histidine-specific amino acid transporter in arabidopsis.

We have identified a new amino acid transporter from the Arabidopsis thaliana expressed sequence tag cDNA collection by functional complementation of a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast shows that it is a high-affinity transporter for both lysine (Lys) and histidine with Michaelis constant values of 175 and 400 microM, respectively. This transporter (LHT1, lysine histidine transporter) has little affinity for arginine when measured directly in uptake experiments or indirectly with substrate competition. The cDNA is 1.7 kb with an open reading frame that codes for a protein with 446 amino acids and a calculated molecular mass of 50.5 kD. Hydropathy analysis shows that LHT1 is an integral membrane protein with 9 to 10 putative membrane-spanning domains. Southern-blot analysis suggests that LHT1 is a single-copy gene in the Arabidopsis genome. RNA gel-blot analysis shows that this transporter is present in all tissues, with the strongest expression in young leaves, flowers, and siliques. Wholemount, in situ hybridization revealed that expression is further localized on the surface of roots in young seedlings and in pollen. Overall, LHT1 belongs to a new class of amino acid transporter that is specific for Lys and histidine, and, given its substrate specificity, it has significant promise as a tool for improving the Lys content of Lys-deficient grains.

Amino acid transport systems required for diazotrophic growth in the cyanobacterium Anabaena sp. strain PCC 7120.

Uptake of 16 amino acids by the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was characterized with regard to kinetic parameters of transport, intracellular accumulation of the transported amino acids, and sensitivity of the transport process to energy metabolism inhibitors. Mutants resistant to certain toxic analogs of some amino acids were isolated that were impaired in amino acid transport. Results obtained in this study, together with those reported previously (A. Herrero and E. Flores, J. Biol. Chem. 265:3931-3935, 1990), suggest that there are at least five amino acid transport systems in strain PCC 7120: one high-affinity, active system for basic amino acids; one low-affinity, passive system for basic amino acids; two high-affinity, active systems with overlapping, but not identical, specificities for neutral amino acids; and one putative system for acidic amino acids. Some of the amino acid transport mutants were impaired in diazotrophic growth. These mutants were unable to develop a normal percentage of heterocysts and normal nitrogenase activity in response to nitrogen stepdown. Putative roles for the amino acid transport systems in uptake of extracellular amino acids, recapture of amino acids that have leaked from the cells, and intercellular transfer of amino acids in the filaments of Anabaena sp. strain PCC 7120 are discussed.

Neurospora mutants affecting polyamine-dependent processes and basic amino acid transport mutants resistant to the polyamine inhibitor, alpha-difluoromethylornithine.

Polyamines (spermidine and spermine) are required by living cells, but their functions are poorly understood. Mutants of Neurospora crassa with enhanced or diminished sensitivity to interference with polyamine synthesis, originally selected to study the regulation of the pathway, were found to have unexpected defects. A group of four non-allelic mutations, causing no interference with polyamine synthesis, each imparted spermidine auxotrophy to a genotype already partially impaired in spermidine synthesis. Strains carrying only the new mutations displayed unconditional delay or weakness at the onset of growth, but grew well thereafter and had a normal or overly active polyamine pathway. These mutants may have defects in vital macromolecular activities that are especially dependent upon the polyamines-activities that have not been identified with certainty in studies to date. Another group of mutants, selected as resistant to the polyamine inhibitor difluoromethylornithine (DFMO), had normal activity and regulation of ornithine decarboxylase, the target of the drug. All but one of thirty mutants were allelic, and were specifically deficient in the basic amino acid permease. This mechanism of DFMO resistance is unprecedented among the many DFMO-resistant cell types of other organisms and demonstrates that DFMO can be used for efficient genetic studies of this transport locus in N. crassa.

Cloning a plant amino acid transporter by functional complementation of a yeast amino acid transport mutant.

Amino acids are transported across the plasma membrane of plant cells by proton-amino acid symports. We report here the successful cloning of a neutral amino acid carrier by functional complementation. A histidine transport deletion mutant of Saccharomyces cerevisiae was transformed with an Arabidopsis thaliana cDNA library constructed in a yeast expression vector. Forty transformants, out of 10(5), allowed growth on a histidine-limiting medium. The acquired ability to grow on low histidine was shown to be strictly dependent on the protein encoded by the expression plasmid. Histidine and alanine transport activity were 10- to 20-fold greater in the transformants. The transport kinetics, inhibitor sensitivity, and substrate specificity match those of neutral system II, a neutral amino acid carrier we previously described in plasma membrane vesicles isolated from leaf tissue. The cDNA insert is 1.7 kb with an open reading frame that codes for a protein containing 486 amino acids with a calculated molecular mass of 52.9 kDa and three sites of potential N-linked glycosylation. Hydropathy analysis of the deduced amino acid sequence suggests this is an integral membrane protein with 10-12 membrane-spanning alpha-helices. Overall, the sequence of this amino acid carrier is not closely related to any other protein sequences in the GenBank data base. Interestingly, however, there are small regions of sequence that exhibit significant levels of similarity with at least seven other integral membrane proteins.

Uptake of galacturonic acid in Erwinia chrysanthemi EC16

Uptake of [14C]galacturonic acid in Erwinia chrysanthemi was found to be stimulated during growth on pectin and its degradation products, saturated digalacturonic acid and galacturonic acid. Cells isolated from macerated potato tissue also showed increased levels of uptake activity for this molecule compared with those showed by glycerol-grown cells. Uptake was found to be an active process, and it displayed saturation kinetics. An Escherichia coli galacturonic acid transport mutant harboring the E. chrysanthemi exuT gene(s) for galacturonic acid uptake was able to transport galacturonic acid but unable to take up the dimer [3H]digalacturonic acid.

Carbohydrate and organic acid composition of effective and ineffective root nodules of Phaseolus vulgaris

Dicarboxylic acid transport mutants of Rhizobium species are usually deficient in their ability to fix atmospheric dinitrogen. We report here a study comparing the physiology of root nodules on Phaseolus vulgaris L. cv. Goldie induced by an effective strain of Rhizobium leguminosarum biovar phaseoli and a C4‐dicarboxylic acid utilization mutant. The mutant, while able to form nodules, was ineffective in N2 fixation. Carbohydrates and organic acids of roots and nodules formed by the 2 strains were monitored at 3‐day intervals from 13 to 34 days after inoculation. Both carbohydrates and organic acids accumulated in ineffective nodules in comparison with the effective nodules. The concentration of malic acid was tenfold higher in ineffective nodules than in effective nodules. Other organic acids, i.e., lactate, malonate, ascorbate and gluconate, were also detected. Lactate and ascorbate were the only other organic acids accumulating in ineffective nodules. The most prevalent carbohydrates found in both types of nodules were sucrose, glucose and fructose. Myo‐inositol was the only cyclitol detected in both types of nodules. Carbohydrates and organic acids were present in lower concentration in roots than in nodules, except for lactate. These compounds were not consistently detected in higher concentration in roots from plants inoculated with the mutant strain, as was the case in nodules.

Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum.

The transport of succinate was studied in bacteroids of an effective, streptomycin-resistant strain (GF160) of Rhizobium leguminosarum. High levels of succinate transport occurred, and the kinetics, specificity, and sensitivity to metabolic inhibitors were similar to those previously described for free-living cells. The symbiotic properties of two transposon (Tn5)-mediated C4-dicarboxylate transport mutants (strains GF31 and GF252) were determined. Strain GF31 formed ineffective nodules, and bacteroids from these nodules showed no succinate transport activity. Strain GF252 formed partially effective nodules, and bacteroids from these nodules showed about 50% of the succinate transport activity of the parent bacteroids. Another dicarboxylic acid transport mutant (Dct-), strain GFS5, isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, formed ineffective nodules. The ability to form ineffective nodules in strains GF31 and GFS5 was shown to correlate with the Dct- phenotype. The data indicate that the presence of a functional C4-dicarboxylic acid transport system is essential for N2 fixation to occur in pea nodules.

Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating.

A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of [3H]proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment.

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.

Characteristics of a transport-deficient mutant ( nap) of Neurospora crassa

nap, previously characterized by Jacobson and Metzenberg as a neutral and acidic amino acid transport mutant, was found in this study to be reduced in transport activity for a variety of metabolites. Analysis of glycoprotein molecules involved in amino acid binding indicates that the deficiency is not at this level. The deficiency appears to be in some common component of all active transport systems.

Suppression of a dicarboxylic acid transport mutant phenotype in Escherichia coli K12

1. 1. Propionate-utilizing mutants of Escherichia coli K12 supress the dicarboxylic transport mutant phenotype; that is, these C 4-acid transport mutants regain the capacity to grow on a dicarboxylic acid as a carbon source. 2. 2. Mutants harboring both the propionate and transport mutations are unable to transport C 4-dicarboxylic acids at low concentrations (1o −5 M), however, they apparently metabolize them at higher concentrations. 3. 3. The propionate mutation acts as a physiological suppressor of a transport defect by causing the simultaneous increase in activity of a number of tricarboxylic acid cycle enzymes concerned with dicarboxylic acid metabolism.

Disruption of an amino acid transport mutant of Neurospora crassa by KCl.

A double amino acid transport-deficient mutant (Pm (-)NB) of Neurospora crassa is shown to be altered in the molecular structure of its cell wall or membrane. This alteration was revealed by a high degree of cellular disruption and cell-cell interaction following extraction by a high molar concentration of KCl.