Concomitant loss of respiratory chain NADH: ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger

Summary Growth, citric acid production and enzymatic activity of the mitochondrial respiratory enzymes of a wild-type and a citric-acid-producing mutant of Aspergillus niger have been compared during fermentation under citric-acid-accumulating and non-accumulating conditions. Under non-accumulating conditions, both strains showed standard growth and no citric acid production. The mutant strain was characterized by delayed onset of growth and lowered cell yield. Under citric-acid-accumulating conditions the wild-type strain exhibited decelerated growth and a maximal citric acid concentration of 12 g l^–1. Reduced, but continuing growth and citric acid production of 32 g l^–1 was observed for the mutant strain. In general, the mutant strain exhibited reduced activity for the proton-pumping respiratory complexes and enhanced activity for the alternative respiratory enzymes. In contrast to the stable activity of complex I in the wild-type strain, this complex was selectively lost in the mutant strain at the onset of citric acid production, while the alternative NADH dehydrogenases were kept at enhanced and constant activity. A possible causal connection between the loss of complex I and citric acid accumulation is discussed. Offsprint requests to: J. Wallrath     

H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate

A mutant of Corynebacterim glutamicum ('Brevibacterium flayum') ATCC14067 with a reduced H+-ATPase activity, F172-8, was obtained as a spontaneous neomycin-resistant mutant. The ATPase activity of strain F172-8 was reduced to about 25% of that of the parental strain. Strain F172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor. It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent. The respiration rate per cell of the mutant also increased to twice as much as that of the parent. However, the growth rate of the mutant was lower than that of the parent. Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid. Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose. Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant. On the other hand, the parent accumulated alpha-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products. It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172-8, because a revertant of strain F172-8, R2-1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent. Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172-8.     

Effects of the tomato pathogen Fusarium oxysporum f. sp. radicis-lycopersici and of the biocontrol bacterium Pseudomonas fluorescens WCS365 on the composition of organic acids and sugars in tomato root exudate

The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bacterial biocontrol strain Pseudomonas fluorescens WCS365, and of both microbes, on the amounts and composition of root exudate components of tomato plants grown in a gnotobiotic stonewool substrate system were studied. Conditions were selected under which introduction of F. oxysporum f. sp. radicis-lycopersici caused severe foot and root rot, whereas inoculation of the seed with P. fluorescens WCS365 decreased the percentage of diseased plants from 96 to 7%. This is a much better disease control level than was observed in potting soil. Analysis of root exudate revealed that the presence of F. oxysporum f. sp. radicis-lycopersici did not alter the total amount of organic acids, but that the amount of citric acid decreased and that of succinic acid increased compared with the nontreated control. In contrast, in the presence of the P. fluorescens biocontrol strain WCS365, the total amount of organic acid increased, mainly due to a strong increase of the amount of citric acid, whereas the amount of succinic acid decreased dramatically. Under biocontrol conditions, when both microbes are present, the content of succinic acid decreased and the level of citric acid was similar to that in the nontreated control. The amount of sugar was approximately half that of the control sample when either one of the microbes was present alone or when both were present. Analysis of the interactions between the two microbes grown together in sterile tomato root exudate showed that WCS365 inhibited multiplication of F. oxysporum f. sp. radicis-lycopersici, whereas the fungus did not affect the number of CFU of the bacterium.     

Stereospecificity of citrate synthetase in relation to glutamate biosynthesis by extracts of Chloropseudomonas ethylicum

The synthesis of citric and glutamic acids by extracts of Chloropseudomonas ethylicum was studied with labeled precursors. When acetyl-coenzyme A-1-(14)C was used as substrate, only 0.1% of the total radioactivity was found in the C-5 position of citric acid; whereas, with oxalacetate-4-(14)C as substrate, 100% of the total radioactivity was found in C-5. These results demonstrated that the Chloropseudomonas citrate synthetase had an absolute stereospecificity, identical to that of the pig heart synthetase. The distribution of radioactivity in the glutamic acid synthesized from acetyl-coenzyme A-1-(14)C was 0% in C-1 and 94.0% in C-5; whereas the glutamic acid formed from oxalacetate-4-(14)C contained 89.6% in C-1 and 0.5% in C-5. This distribution is entirely consistent with the biosynthesis of glutamic acid from citric acid via aconitase, d(s)-isocitrate, and l-glutamate dehydrogenases. The presence of l-glutamate dehydrogenase in extracts was demonstrated. The stereospecificity of the citrate synthetase and the pattern of glutamate labeling further establish that the aconitase of Chloropseudomonas is completely stereospecific.     

Metabolism of glutamic acid in a mutant of Escherichia coli

Vender, Joyce (Indiana University, Bloomington), Kunthala Jayaraman, and H. V. Rickenberg. Metabolism of glutamic acid in a mutant of Escherichia coli. J. Bacteriol. 90:1304-1307. 1965.-A mutant strain of Escherichia coli W1485 was selected for its ability to utilize glutamic acid as the sole source of carbon. Growth of the mutant on glutamic acid led to the repression of glutamic acid dehydrogenase formation. The mutant differed from the wild-type strain in that glutamic decarboxylase activity was absent from the mutant under conditions of growth which supported the formation of this enzyme in the parent strain. Evidence is presented which suggests that loss of the decarboxylase activity results in the acquisition of the ability to utilize glutamic acid as sole source of carbon; a pathway of glutamate utilization via transamination is proposed.     

Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).     

Characteristics of modulators and substrates binding to rat liver adenylate kinase.

The binding of adenine nucleotides to liver adenylate kinase was dependent on Mg2+ ions. Citric acid enhanced the binding of all metal-chelated radioactive nucleotides and indicated two observable binding sites for Mg3H-ADP and Mg3-ATP and one-half binding site for Mg3H-AMP. Two binding sites of Mg3H-ADP and one binding site for Mg3H-ATP were also observed in the absence of citric acid. Stoichiometric binding of 14C-citric acid to liver adenylate kinase varied with additions of different nucleotides. AMP prevented whereas ADP and ATP enhanced the binding of 14C-citric acid.     

Effects of Mutations and Growth Conditions on Lipid Synthesis in Neurospora crassa

A morphological mutant (col-2) of Neurospora, which is partially deficient in glucose-6-phosphate dehydrogenase (G-6-PD) activity and has lower levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH), accumulated three-fold more triglycerides during log-phase growth than the wild-type strain. Increased lipid deposition was not found in other strains that included slow-growing morphological mutants, NADPH-deficient strains, G-6-PD-deficient mutants, wild-type revertants from col-2, and a cel, col-2 double mutant. The cel, col-2 strain was supplemented with an exogenous source of fatty acids because it cannot synthesize these lipid moieties. The observed normal lipid content of this strain suggests that the lipid deposition in col-2 on glucose is due to an overstimulation of fatty acid synthesis and not a deficiency in fatty acid breakdown. The neutral lipid levels in both wild type and col-2 were decreased to identical levels when grown on glutamate as a carbon source. This effect was not due to changes in glutamic dehydrogenase levels. The omission of citrate from the glutamate medium reduced wild-type neutral lipid levels even further, but had no effect on col-2. The variations with time in the neutral lipid levels of col-2 upon changes in these carbon sources are presented, as well as a discussion of the possible types of regulatory effects unique to the col-2 mutation which might affect fatty acid synthesis.     

Phage Growth and Deoxyribonucleic Acid Synthesis in Escherichia coli Infected by a Thymine-Requiring Bacteriophage.

Mathews, Christopher K. (Yale University, New Haven, Conn.). Phage growth and deoxyribonucleic acid synthesis in Escherichia coli infected by a thymine-requiring bacteriophage. J. Bacteriol. 90:648-652. 1965.-Cultures of Escherichia coli B infected with a mutant strain of phage T4 which cannot induce the formation of thymidylate synthetase produce deoxyribonucleic acid (DNA) at about two-thirds the rate of cultures infected with the parent strain. Under certain conditions the yield of viable phage observed with the mutant is one-third of that brought about by the wild-type strain. Addition of thymine increases both DNA synthesis and phage production in cells infected by the mutant. It is suggested that the ability to induce thymidylate synthetase formation in infected cells confers a selective advantage on the wild-type strain.     

Citric acid in tomato plant roots and its effect on cadmium uptake and distribution

Experiments were carried out to investigate the effects of root citric acid on uptake and initial distribution of cadmium (Cd) in tomato plants (Lycopersicon esculentum, cv. Tiny Tim). Cd was measured by γ-spectrometry, using ¹¹⁵Cd spikes. Citric acid was measured by UV-detection, and, after spiking with ¹⁴C-citric acid, by β-spectrometry. Cd was applied for 48 h periods, in control experiments, in the presence of citric acid, and after 24 h plant pre-incubation with citric acid. Pre-incubation resulted in two-fold increases in fast-exchangeable amounts of root citric acid, as measured by the presence of citric acid in xylem exudates of decapitated and pressurized roots. Simultaneous application of Cd and citric acid did not change Cd accumulation in total plants and in the roots, nor did any significant change occur with respect to Cd root-to-shoot transport, and Cd concentrations in shoot tissues and xylem fluid. After citric acid pre-incubation, total plant uptake of Cd increased twofold, without any significant change in Cd accumulation in the roots. Cd root-to-shoot transport was increased 5–6 fold, and Cd concentrations in shoot tissues and xylem fluid were increased 6–8 fold. Speciation calculations indicated that, under the conditions applied, xylem Cd may be, at least partly, complexed in citric acid. A C Borstlap Section editor     

Influence of the glycolytic rate on production of citric acid and oxalic acid by Aspergillus niger in solid state fermentation

A study was performed to understand the physiology and biochemical mechanism of citric acid accumulation during solid state fermentation of sweet potato using Aspergillus niger Yang No.2. A low citrate-producing mutant was isolated followed by a comparative study of the fermentation process and selected physiological and biochemical parameters. In contrast with the parent strain, the mutant strain displayed lower concentrations, yields and production rates of citric acid, accompanied by higher concentrations, yields and production rates of oxalic acid. In addition, the mutant utilized starch at a lower rate although higher concentrations of free glucose accumulated in the cultures. Biochemical analyses revealed lower rates of glucose uptake and hexokinase activity of the mutant strain in comparison with the parent strain. It is proposed that, in common with submerged fermentation, over-production of citric acid in solid state fermentation is related to an increased glucose flux through glycolysis. At low glucose fluxes, oxalic acid is accumulated.     

碱胁迫下耐碱植物星星草体内柠檬酸特异积累现象(英)

对碱胁迫 (0 - 175mmol/LNa2 CO3 )下星星草 (Puccinelliatenuiflora (Griseb .)Scribn .etMerr.)体内柠檬酸的积累规律及其相关胁变指标进行分析测定。实验结果证明 :积累柠檬酸是星星草对碱胁迫特有的生理反应。盐胁迫(0 - 4 0 0mmol/LNaCl)下 ,柠檬酸含量反而稍有下降。柠檬酸积累量随碱胁迫强度增大而增大 ,低胁迫强度时积累量上升缓慢 ,当胁迫强度大于 10 0mmol/LNa2 CO3 时 ,积累量明显上升。柠檬酸积累与胁迫时间之间呈直角曲线关系 ,一定胁迫强度下胁迫 4h后即可测出柠檬酸含量明显上升 ,约 4 8h后渐趋最大值。碱胁迫 14 4h后星星草各部位中柠檬酸含量从高到低的顺序依次是老叶、成熟叶、老叶鞘、幼叶鞘、幼茎、老茎和幼叶。成熟叶中柠檬酸随碱胁迫强度增大而逐渐上升 ,老叶和叶鞘中的柠檬酸在碱胁迫强度大于 12 5mmol/L后急剧上升 ,茎中柠檬酸含量无明显增高 ,幼叶中柠檬酸含量基本不变。实验证明 ,碱胁迫下积累的主要是柠檬酸 ,其他有机酸无明显变化。     

Glutamic Acid Production in Biotin-rich Media by Temperature-sensitive Mutants of Brevibacterium lactofermentum,a Novel Fermentation Process

Temperature-sensitive mutants were derived from Brevibacterium lactofermentum strain 2256 in a search for mutants which would produce a large amount of L-glutamic acid in biotin- rich media at the nonpermissive temperature. A total of 159 mutant strains was selected which showed adequate growth at 30°C but showed little or no growth at 37°C on minimal medium. Twenty of these were found to produce glutamic acid in a biotin-rich medium after a temperature shift from 30°C to 37°C, while the wild-type strain 2256 did not produce it under the same cultural condition.

One of the typical mutant strains, Ts-88, produced approximately 2g/dl of glutamic acid from beet molasses (the yield > 55%) in the presence of 33 µg/liter of biotin when tempera- , ture was shifted from 30°C to 40°C during the cultivation. It was concluded that, by controlling only temperature during fermentation, glutamic acid production could be realized in media containing biotin-rich natural carbon sources, without any chemical control such as the addition of expensive surface-active agents or antibiotics. Characteristics and merits of the novel fermentation process are discussed.     

Nitrogen regulation of arginase in Neurospora crassa.

The final products of the arginine catabolism that can be utilized as a nitrogen source in Neurospora crassa are ammonium, glutamic acid, and glutamine. The effect of these compounds on arginase induction by arginine was studied. In wild-type strain 74-A, induction by arginine was almost completely repressed by glutamic acid plus ammonium, whereas ammonium or glutamic acid alone had only moderate effects. Arginine products of catabolism also repressed arginase induction. A mutant, ure-1, which lacks urease activity, hyperinduced its arginase with arginine as a nitrogen source. The addition of either ammonium or glutamine produced effects similar to those in the wild-type strain. The effect of ammonium on arginase induction is mediated through its conversion into glutamine. This was demonstrated in mutant am-1, which lacks L-glutamate dehydrogenase activity. In this mutant, the effect of glutamic acid was reduced, and, with ammonium, it was completely lost. The addition of glutamine or glutamic acid plus ammonium to this strain decreased by threefold the induction of arginase by arginine. Proline, a final product of arginine catabolism, competitively inhibited arginase activity. This effect and the repression of arginase by glutamine are examples of negative modulation of the first enzyme in a catabolic pathway by its final products.     

Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032

To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum.     

Transport of Aminophosphonic Acids in Lactobacillus plantarum and Streptococcus faecalis

Aminophosphonic acids analogous to glutamic acid, aspartic acid, alanine, and valine were actively accumulated by Lactobacillus plantarum. Uptake was dependent on the availability of glucose and, in all cases, the estimated intracellular concentrations substantially exceeded extracellular levels. During uptake, there was little metabolism of tritiated 2-amino-3-phosphonopropionic acid (APP), the aspartic acid analogue, and a negligible incorporation of isotope from this substance into the nucleic acid, lipid, protein, or cell wall fractions of the cell. Competition studies with APP indicated that its transport in L. plantarum and in Streptococcus faecalis was antagonized only by structurally related compounds such as glutamic, aspartic, and cysteic acids. Kinetic studies showed that APP was taken up by a single catalytic system in S. faecalis. A mutant strain of this organism which lacks one of two kinetically distinguishable dicarboxylic amino acid transport systems failed to accumulate measurable amounts of APP. These experiments indicate that the aminophosphonic acids are accumulated by the amino acid transport systems in these bacteria with minimal metabolic changes.     

Preliminary Screening of Microorganisms Producing Succinic Acid from n-Paraffin

Screening tests in search for microorganisms capable of producing succinic acid from n-paraffin were carried out. Most of the microorganisms that accumulated succinic acid in culture broth when incubated in the media containing super heavy n-paraffin as the carbon source were found to belong to the genus Candida. The largest quantity of succinic acid production from n-paraffin, 4160 μ/ml, was obtained with a strain, Candida brumptii IFO 0731.     

碱胁迫下耐碱植物星星草体内柠檬酸特异积累现象

对碱胁迫(0-175 mmol/L Na2CO3)下星星草(Puccinellia tenuiflora (Griseb.) Scribn.et Merr.)体内柠檬酸的积累规律及其相关胁变指标进行分析测定.实验结果证明:积累柠檬酸是星星草对碱胁迫特有的生理反应.盐胁迫(0-400 mmol/L NaCl)下,柠檬酸含量反而稍有下降.柠檬酸积累量随碱胁迫强度增大而增大,低胁迫强度时积累量上升缓慢,当胁迫强度大于100 mmol/L Na2CO3时,积累量明显上升.柠檬酸积累与胁迫时间之间呈直角曲线关系,一定胁迫强度下胁迫4 h后即可测出柠檬酸含量明显上升,约48 h后渐趋最大值.碱胁迫144 h后星星草各部位中柠檬酸含量从高到低的顺序依次是老叶、成熟叶、老叶鞘、幼叶鞘、幼茎、老茎和幼叶.成熟叶中柠檬酸随碱胁迫强度增大而逐渐上升,老叶和叶鞘中的柠檬酸在碱胁迫强度大于125 mmol/L后急剧上升,茎中柠檬酸含量无明显增高,幼叶中柠檬酸含量基本不变.实验证明,碱胁迫下积累的主要是柠檬酸,其他有机酸无明显变化.     

Control of Uridine Diphosphate-Glucose Dehydrogenase Synthesis and Uridine Diphosphate-Glucuronic Acid Accumulation by a Regulator Gene Mutation in Escherichia coli K-12

Uridine diphosphate (UDP)-glucose dehydrogenase, the enzyme that converts UDP-glucose to UDP-glucuronic acid, was derepressed in a mucoid (capR9) strain of Escherichia coli K-12 and repressed in a nonmucoid (capR(+)) strain. A nonmucoid mutant (strain MC 152; capR9 non-2) derived from the mucoid strain accumulated large quantities of nucleotides. Among these nucleotides, UDP-glucuronic acid was identified as well as guanosine triphosphate and an adenosine diphosphate-sugar. UDP-glucose dehydrogenase was still derepressed in strain MC 152. When the nonmucoid mutant was transduced to the wild-type state for this regulator gene (capR(+)), the transductant was found to accumulate less total nucleotides, and the accumulation of UDP-glucuronic acid was abolished. UDP-glucose dehydrogenase was repressed in the capR(+)non-2 strain but not to the same extent that it was in the capR(+) strain.     

Detection of Glycollic Acid in Etiolated Barley Shoots

A modified method for carboxylic acid analysis has been developedin order to study the metabolism of glycollic acid in barleysap. Glycollic, malic, citric, malonic, succinic, and fumaric acidshave been detected in alcoholic extracts from etiolated barleyshoots, and the amounts present roughly estimated.