Studies of partially repressed mutants at the tamA and areA loci in Aspergillus nidulans

Summary Mutants, designated tamA ^r, have been isolated on the basis of simultaneous resistance to toxic analogues thiourea, aspartate hydroxamate and chlorate with L-alanine as the sole nitrogen source. tamA ^r mutants are also resistant to methylammonium. This resistance of tamA ^r mutants is correlated with partially repressed activity of a number of enzyme and transport systems regulated by ammonium. Furthermore, tamA ^r mutants have low NADP-glutamate dehydrogenase (NADP-GDH) activity and also efflux ammonium under certain growth conditions.Mutants at the areA locus (areA ^r) have also been isolated on the basis of resistance to these analogues, with nitrate or L-aspartate as the nitrogen source. These, similar to tamA ^r lesions, result in resistance to methylammonium and are partially repressed for ammonium repressible systems, but in contrast to tamA ^r, areA ^r alleles have wild-type NADP-GDH activity and normal ammonium efflux. tamA ^r and areA ^r mutants grow as wild type on all nitrogen or carbon sources tested, are recessive, and appear to be epistatic to all other mutations (gdhA1, meaA8 and meaB6) which result in derepressed levels of ammonium regulated system. Whereas tamA ^r and areA ^r phenotypes are additive, tamA ^r is epistatic to areA ^d phenotype.     

Cis-dominant regulatory mutations affecting the expression of GABA permease in Aspergllus nidulans

Summary In Aspergillus nidulans expression of the gabA gene, the probable structural gene for the -amino-n-butyrate (GABA) permease, is controlled by induction, via the intA gene, ammonium repression, mediated by the areA gene, and probably carbon catabolite repression. Regulatory mutations, tightly linked to gabA, were selected by reverting an areA-2 strain on GABA as nitrogen source. These mutations, gabI-1, gabI-2, and gabI-3 result in increased gabA expression and are cis-dominant in their effects on the gabA gene. Mapping data show that the regulatory mutations map on one side of all gabA^- alleles tested.     

Pleiotropic mutants of Aspergillus nidulans altered in carbon metabolism.

Summary Mutants altered in carbon catabolite regulation have been isolated by selecting for mutants of theareA217 strain capable of using acetamide as the sole nitrogen source in the presence of sucrose. In addition tocreA mutants described previously by Arst and Cove, strains with mutations in two new genes,creB andcreC, have been found. ThecreB andcreC mutants grow poorly on some sole carbon sources and have low levels of some enzymes of carbon catabolism e.g. -galactosidase and D-quinate dehydrogenase. ThecreB andcreC mutants are hypersitive to fluoroacetate, fluoroacetamide and allyl alcohol in the presence of glucose or sucrose but not glycerol; and the enzymes, acetamidase, and alcohol dehydrogenase, are less sensitive to carbon catabolite repression than the wild-type strain. Extracellular protease and -glucosidase enzyme activities are elevated increB andcreC mutants, while L-proline and L-glutamate uptake capacities are lower in both the presence and absence of glucose. Interactions betweencreA, B and C mutations have been investigated in double mutants, and the dominance properties ofcreB andcreC mutants determined. The results indicate that thecreB andcreC genes may have a regulatory role in the control of carbon catabolism.     

d-Alanine dehydrogenase

Pseudomonas aeruginosa PA01 was found to utilise both thed- andl-isomers of -alanine and also -alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation ofd-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation ofl-alanine required its conversion to the directly oxidisabled-form by a soluble racemase; (iii) utilisation of -alanine, likel-alanine, involves both the racemase andd-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity, of which is still speculative; (iv)P. aeruginosa, likeEscherichia coli, appears to take upd-alanine andl-alanine by means of two specific permeases.Abbreviation DCPIP 2,6-dichlorophenol-indophenol     

A mutation in Aspergillus nidulans which affects the regulation of nitrite reductase and is tightly linked to its structural gene.

Summary The selection of nis-5, a mutation which is tightly linked to the structural genes for nitrate reductase (niaD) and nitrite reductase (niiA) but which only affects nitrite reductase activities, is described. nis-5 single mutants have only 40% of the wild type activity of nitrite reductase after induction by nitrate and, for this reason, grow poorly on nitrate and nitrite. Nitrate reductase activity is not affected, and nis-5 is shown to complement with a niaD^- mutation but not with a niiA^- mutation.When grown without inducer, nis-5 strains have higher than the non-induced wild type activity of nitrite reductase. This low, constitutive activity is insensitive to repression by ammonium. These facts explain why the nis-5 mutation weakly suppresses many nirA^- and areA^r mutations for utilization of nitrite.Three of the possible explanations of this unusual phenotype are considered. Studies of nitrite reductase in cell-free extracts provided no evidence for the already unlikely possibility that nis-5 is a structural gene mutation resulting in the observed phenotype because of alteration in the catalytic activity and/or stability of the nitrite reductase.A more plausible explanation is that it defines a receptor site for either the nirA gene product and/or the areA gene product. However, no evidence for this has yet been obtained from a study of double mutants carrying nis-5 and areA or nirA mutations.A third possibility is that nis-5 creates a new, but inefficient promoter or initiator, which is not subject to the normal control systems (and therefore causes constitutive, deprepressed synthesis) but whose physical presence reduces maximal enzyme synthesis. The presence of a translocation in nis-5 strains suggests a means by which niiA could come to be under the control of another promoter/initiator.     

Studies on Glucose Isomerase of Bacteria

A bacterial strain, HN-500, having an activity of d-glucose isomerization was newly isolated from soil, and was identified to be similar to Escherichia intermedia (Werkman and Gillen) Vaughn and Levine. The strain, grown on wide varieties of carbon sources, shows definitely d-glucose isomerizing activity in the presence of arsenate. d-Fructose formed in reaction mixture was identified by paper chromatography and was isolated in crystalline form from calcium-fructose complex. In order to increase the production of d-glucose isomerase, d-glucose and ammonium nitrogen were effective carbon and nitrogen sources, respectively, but none of the metallic ions tested were effective, furthermore manganese, ferrous and ferric ions present mOre than 10^-5m in growth medium fully repressed the enzyme formation. The cells grown on carbon sources other than d-xylose showed no activity of d-xylose isomerase.     

Amide metabolism of Chlamydomonas reinhardi

Chlamydomonas reinhardi can utilise the lower aliphatic amides (C₁–C₄) as nitrogen sources. Of these only acetamide can serve as a sole carbon source. The acetamide analogue F-acetamide kills cells after conversion to F-acetate and F-citrate. This conversion is controlled by exogenous ammonia and, in part, acetate levels. Only one enzyme and one active site are involved in acetamidase function. Enzymatic analysis indicates an increased substrate range as compared to the growth — supported range, indicating uptake, toxicity or metabolic control restrictions.Abbreviations TCA trichloroacetic acid - TAP tris-acetate-phosphate medium - MIC mimmum inhibitory concentration - BSA bovine serum albumin     

The genetic analysis of regulation of amidase synthesis in Aspergillus nidulans. I. Mutants able to utilize acrylamide

Summary Aspergillus nidulans uses an acetamidase enzyme to grow on acetamide as a carbon or as a nitrogen source. Acrylamide is a substrate for the enzyme but does not induce its synthesis. Mutants capable of growing on acrylamide as a nitrogen source have been isolated. Two classes of mutant have been found —amdR ^c mutants on linkage group II andamdT ^c on linkage group III.amdR ^c mutants produce high constitutive acetamidase levels. The enzyme is still inducible by amides, but to a lesser extent than wild type, and is still subject to repression by ammonia and by carbon metabolites derived from glucose.amdR ^c mutants are semi-dominant to the wild type allele in heterozygous, diploids. TheamdT ^c mutant is not subject to carbon metabolite repression, of the acetamidase. The enzyme is inducible by amides and repressible by ammonia. TheamdT ^c mutation also results in reduced ability to grow on formamide as a nitrogen source and to lowered levels of a second amidase enzyme.amdT ^c is semi-dominant in heterozygous diploids.     

The regulation of hexokinase and phosphoglucomutase activity in Aspergillus nidulans.

Summary The levels of glucose-6-phosphate and 6-phosphogluconate dehydrogenase in wildtype cells of Aspergillus nidulans varied with the carbon and nitrogen source. In general, hexokinase activity did not vary with carbon or nitrogen source. The ammonium derepressed mutant amrA1 had only 50% of the wildtype level of hexokinase. Phosphoglucomutase activity was low in wildtype cells grown with nitrate, but high in cells grown with ammonium when glucose was the carbon source. A non-inducible mutant, nirA ^-1, in the regulatory gene for nitrate reductase, had high phosphoglucomutase activity when grown with nitrate or ammonium. A constitutive mutant nirA ^c1, in the regulatory gene for nitrate reductase had low phosphoglucomutase activity when grown with nitrate or ammonium. The mutants nir ^-1 and nirA ^c1 are recessive and semi-dominant respectively for abnormal phosphoglucomutase activity.     

A yeast mutant with glucose-resistant formation of mitochondrial enzymes

Summary Yeast mutants with glucose-insensitive formation of mitochondrial enzymes were isolated starting with a strain completely lacking alcohol dehydrogenase activity. The mutations could uniquely be attributed to a single nuclear gene, designated CCR80. They were largely dominant. Glucose-resistant enzyme formation was most prominent with regard to mitochondrial enzymes succinate dehydrogenase and NADH: cytochrome c oxidoreductase. The effect of CCR80 ^r mutations was rather small but significant on the gluconeogenetic enzymes isocitrate lyase, malate synthase and fructose-1,6-bisphosphatase and on invertase synthesis. The repressive effect of maltose in CCR80 ^r mutants was also reduced showing that glucose-resistance is not caused by a mere hexose uptake defect. This regulatory disorders were not accompanied by reduced levels of glycolytic enzymes or drastically altered levels of glycolytic intermediates.Aerobic fermentation of glucose was almost completely inhibited in the mutants; anaerobic glucose degradation was reduced but not completely abolished. Therefore, the mutants appear to be altered in the regulation of glycolysis. A largely glucose-resistant synthesis of respiratory enzymes is obviously a corollary of this alteration.     

Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe

Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamineoxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as K _m values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.     

Altered S5 and S20 ribosomal proteins in revertants of an alanyl-tRNA synthetase mutant of Escherichia coli

Summary An active transport system specific for ammonium and methylammonium is decribed in wild type cells of Aspergillus nidulans. This system has a Km of less than 5x10^-5 M for ammonium as measured by the uptake of ¹⁵NH⁺ ₄ and a Km of 2x10^-5 M and apparent Vmax of 11 nanomoles/min/mg dry weight for methylammonium, by the uptake of ¹⁴C methylammonium. The system concentrates methylammonium at least 120-fold and is probably regulated by the concentration of internal ammonium.Cells of the mutant strain DER-3 possess a reduced rate of ammonium and methylammonium transport under all conditions tested. DER-3 is a double mutant, one mutation being allelic with meaA8 and designated meaA21, the other is unlinked to meaA and designated mod meaA. The heterozygous diploid DER3/+ has wild type transport, indicating that the mutations are recessive. Cells of the mutant strain amrA1 have impaired transport of ammonium and methylammonium, but only under some conditions. amrA1 is recessive. The possible defects of these mutants are discussed.     

Improvement of pristinamycin-producing Streptomyces pristinaespiralis by rational screening

Summary According to the biosynthetic pathway of pristinamycin, a rational selection procedure with u.v. mutation was performed to obtain a high pristinamycin-producing strain. Aminoacetic acid-resistant mutants (AA^r), valine hydroxamate-resistant mutants (VH^r), kitasamycin-resistant mutants (KTM^r) and 2-deoxy-D-glucose-resistant mutants (DOG^r) were selected, successively. A strain Streptomyces pristinaespiralis 12–55 with AA^r, Val^r, KTM^r, and DOG^r was obtained, and its production of pristinamycin reached 3000 u/ml which is 100 times higher than that of the parent strain S. pristinaespiralis ATCC 25486. It is inferred that S. pristinaespiralis 12–55 can alleviate catabolite repression caused by carbon sources, provide more acetic acid and valine for pristinamycin biosynthesis and increase its resistance to pristinamycin produced by itself, all of which are favorable for pristinamycin production. The subculture experiments indicated that the hereditary character of high productivity of S. pristinaespiralis 12–55 is stable. The pristinamycin production of S. pristinaespiralis 12–55 in a 15-l fermentor could reach 3010 u/ml after a 56 h batch fermentation.     

The effect of<Emphasis Type="Italic"> pfl</Emphasis> gene knockout on the metabolism for optically pure <Emphasis Type="SmallCaps">d</Emphasis>-lactate production by<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The effect of gene knockout on metabolism in the pflA^–, pflB^–, pflC^–, and pflD^– mutants of Escherichia coli was investigated. Batch cultivations of the pfl ^– mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA^– and pflB^– mutants, but not pflC^– and pflD^– mutants, produced large amounts of d-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD⁺ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA^– and pflB^– mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD⁺ ratio in pfl ^– mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.     

Glucose repression in Streptomyces coelicolor A3(2): a likely regulatory role for glucose kinase

The glucose kinase gene (glkA-ORF3) of Streptomyces coelicolor A3(2) plays an essential role in glucose utilisation and in glucose repression of a variety of genes involved in the utilisation of alternative carbon sources. These genes include dagA, which encodes an extracellular agarase that permits agar utilisation. Suppressor mutants of glkA-ORF3 deletion strains capable of utilising glucose (Glc⁺) arise at a frequency of about 10^–5 on prolonged incubation. The Glc⁺ phenotype of the mutants is reversible (at a frequency of about 10^–3) and reflects either the activation of a normally silent glucose kinase gene or the modification of an existing sugar kinase. Although the level of glucose kinase activity in the Glc⁺ supressor mutants is similar to that in the glkA ⁺ parental strain, glucose repression of dagA remains defective. Expression of the glucose kinase gene of Zymomonas mobilis in glkA-ORF3 mutants restored glucose utilisation, but not glucose repression of dagA. Over-expression of glkA-ORF3 on a high-copy-number plasmid failed to restore glucose repression of dagA in glkA-ORF3 mutants and led to loss of glucose repression of dagA in a glkA ⁺ strain. These results suggest that glucose phosphorylation itself is not sufficient for glucose repression and that glkA-ORF3 plays a specific regulatory role in triggering glucose repression in S. coelicolor A3(2).     

Biodegradation of chlorinated paraffins and long-chain chloroalkanes by Rhodococcus sp. S45-1

A strain of the genus Rhodococcus, designated isolate S45-1, was isolated from an environmental water sample by enrichment, using the chlorinated paraffin Cereclor S45 as the sole carbon and energy source. This is the first report of microbial utilisation of chlorinated paraffins as sole source of carbon and energy. Biochemical studies of isolate S45-1 revealed little similarity with other Rhodococcus species. Isolate S45-1 was able to utilise 1-chloroalkanes of chain-length 12–18C as sole source of carbon and energy. Gas chromatography-mass spectrometry of the reaction medium indicated that γ-butyrolactone was formed as a product of 1-chlorotetradecane metabolism.     

Growth and nitrate uptake properties of plants grown at different relative rates of nitrogen supply. II. Activity and affinity of the nitrate uptake system in Pisum and Lemna in relation to nitrogen availability and nitrogen demand

Abstract Net nitrate uptake rates were measured and the kinetics calculated in non-nodulated Pisum sativum L. cv. Marma and Lemna gibba L. adapted to constant relative rates of nitrate-N additions (R_A), ranging from 0.03 to 0.27 d^?1 for Pisum and from 0.05 to 0.40 d^?1 for Lemna, V_max of net nitrate uptake (measured in the range 10 to 100 mmol m^?3 nitrate, i.e. ‘system I’) increased with R_A in the growth limiting range but decreased when R_A exceeded the relative growth rate (RGR), K_m was not significantly related to changes in R_A. On the basis of previous ¹³N-flux experiments, it is concluded that the differences in V_max at growth limiting R_A are attributable to differences in influx rates. Linear relationships between V_max and tissue nitrogen concentrations were obtained in the growth limiting range for both species, and extrapolated intercepts relate well with the previously defined minimal nitrogen concentrations for plant growth (Oscarson, Ingemarsson & Larsson, 1989). Analysis of V_max for net nitrate uptake on intact plant basis in relation to nitrogen demand during stable, nitrogen limited, growth shows an increased overcapacity at lower R_A values in both species, which is largely explained by the increased relative root size at low R_A. A balancing nitrate concentration, defined as the steady state concentration needed to sustain the relative rate of increase in plant nitrogen (R_N), predicted by R_A, was calculated for both species. In the growth limiting range, this value ranges from 3.5 mmol m^?3 (R_A 0.03 d^?1) to 44 mmol m^?3 (R_A 0.21 d^?1) for Pisum and from 0.2 mmol m^?3 (R_A 0.05 d^?1) to 5.4 mmol m^?3 (R_A 0.03 d^?1) for Lemna. It is suggested that this value can be used as a unifying measure of the affinity for nitrate, integrating the performance of the nitrate uptake system with nitrate flux and long term growth and demand for nitrogen.     

Pleiotropic mutants affecting the control of nitrogen metabolism in Aspergillus nidulans

Summary Mutants of Aspergillus nidulans with lesions in gene amdT are pleiotropically affected in their ability to utilize a wide variety of nitrogen sources in the presence of glucose. Ability to utilize a number of these compounds as sole sources of carbon and nitrogen is not altered. One of these mutants, amdT102, has properties consistent with it being derepressed for glucose repression of the utilization of most (but not all) nitrogen sources. The amdT102 mutant can grow strongly on histidine, lysine and cystine as sole nitrogen sources while the wild type strain grows extremely poorly on these amino acids. Similar but less extreme effects apply to many other nitrogen sources. The amdT19 mutant is unable to utilize most nitrogen sources in the presence of glucose, suggesting that it is subject to greatly increased repression of nitrogen source utilization. The amdT mutants are not affected in their ability to use many compounds as sole carbon sources. Carbon sources other than glucose also affect utilization of nitrogen sources in the amdT mutants.     

Poor transformability with nov and ery donor DNA of some mitomycin-C-sensitive strains of Haemophilus influenzae

Three mitomycin-C-sensitive (MC^s) strains of Haemophilus influenzae, being poorly transformable with DNA carrying the antibiotic resistence markers nov^r and ery^r, were further investigated to determine the cause of their poor transformability. After being genetically integrated into the mutant-recipient genome the donor marker is replicated at the same rate as in the wild type, indicating that recombination in the mutant strains is normal. In the mutants, designated T^d (transformation-deficient), the poor transformability for the nov^r and ery^r markers is due to the lack of phenotypic expression of the markers, because the strains are killed by concentrations of antibiotics normally used to select for nov^r and ery^r transformants. Since the strains exhibit extreme sensitivity both to deoxycholate and osmotic shock in the presence of EDTA, the increased sensitivity to antibiotics (including mitomycin-C) is probably caused by a change in the cell envelope. Although recombination in the mutant strains proceeds normally, the T^d mutation nevertheless decreases both the rate of inactivation and of integration of donor DNA.