Overexpression of <Emphasis Type="Italic">Citrus junos</Emphasis> mitochondrial citrate synthase gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> confers aluminum tolerance

Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.     

Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture

Summary The effects of citrate on diacetyl, acetoin and 2,3-butylene glycol (2,3-BG) production by Leuconostoc mesenteroides subsp. cremoris grown in continuous culture at pH 5.2 were studied. In glucose alone end-product production agreed with the theoretical stoichiometry. In the presence of citrate, lactate and acetate production was higher than the theoretical stoichiometry from glucose. Lactate production was constant when the initial citrate concentration was increased whereas ethanol production strongly decreased. In the absence of citrate, citrate lyase (CL) exhibited weak activity. Diacetyl reductase (DR) and acetoin reductase (AR) exhibited basal activity. When varying citrate concentrations ranging from 10 to 75 mm were added to glucose broth, DR, AR, lactate dehydrogenase, NADH oxidase and alcohol dehydrogenase decreased as the initial citrate concentration increased suggesting that they were partly repressed by citrate. In contrast, CL increased and the specific citrate utilization rate also increased in the same way, indicating no saturation of the first step of citrate metabolism. Acetate kinase (AK) was slightly higher in the presence of citrate and increased when the initial citrate concentration increased. This result was correlated with an increase of acetate from the acetyl phosphate pathway. More ATP was produced in the presence of citrate, which could explain the increase in biomass formation. Citrate bioconversion into diacetyl, acetoin and 2,3-BG increased as the initial citrate increased. Correspondence to: C. Diviès     

Promotion of Citrate Transport of the Transport-negative Mutants of Aerobacter cloacae

The “citrate transport-enhancing factor” obtained from Aerobacter cloacae did stimulate uptake of radioactive citrate by Escherichia coli, having an intrinsic barrier against citrate permeation. In order to prove function of the factor in the cells of Aerobacter, citrate transport-negative mutants of A. cloacae were isolated. These mutants were found to be lacking in the factor. Addition of the factor to these mutants resulted in stimulation of uptake of citrate. These results evidenced that the factor played an essential role in the citrate transport system of A. cloacae.     

Distribution of the ability for citrate utilization amongst Clostridia

44 species of the genus Clostridium were examined with regard to their ability to grow on citrate. In addition to Clostridium sphenoides, a known citrate utilizer, the following species were found to utilize citrate: C. sporosphaeroides, C. symbiosum, C. rectum, C. indolis, C. subterminale and C. sporogenes. The major products formed from citrate were acetate, ethanol and carbon dioxide (not measured). Minor products were butyrate, butanol, acetone and acetoin.The enzyme citrate lyase was detectable in cell extracts of C. sporosphaeroides and C. symbiosum using an optical assay. Evidence for the presence of this enzyme in the other species was obtained in immunological experiments and in experiments with [1,5-¹⁴C]citrate.     

Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression

In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium-dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ-, β-, and α-subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated M_r 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC-expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5-phosphoribosyi-dephospho-acetyl-coenzyme A which is covalently bound to serine-14 of the acyl carrier protein (λ-subunit). CitC was thus identified as acetate:SH-citrate lyase ligase. The function of the gene product derived from citG (M_r 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl-coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo-citrate lyase.     

Characterization of citrate lyase from Clostridium sporosphaeroides

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form.     

Synergistic iron reduction and citrate dissimilation by Shewanella alga and Aeromonas veronii

Two bacterial isolates from Great Bay Estuary, New Hampshire, in co-culture carried out anaerobic dissimilation of citric acid with Fe(III) as the terminal electron acceptor. Neither isolate oxidized citrate with Fe(III) anaerobically in axenic culture. The Fe(III) reducer, Shewanella alga strain BrY, did not grow anaerobically with citrate as an energy source. The citrate utilizer, Aeromonas veronii, did not reduce iron axenically with a variety of electron donors including citrate. The onset of iron reduction by the co-culture occurred after initiation of citrate dissimilation and just prior to initiation of growth by either organism (as measured by viable plate counts). Anaerobic culture growth rates and final cell densities of each bacterial strain were greater in co-culture than in axenic cultures. By 48 h of growth, the co-culture had consumed 27 mM citrate as compared with 12 mM dissimilated by the axenic culture of A. veronii. By 48 h the co-culture produced half as much formate (6 mM) and twice as much acetate (40 mM) as did A. veronii grown axenically (12 mM and 20 mM, respectively). Formate produced from citrate by A. veronii appeared to have supported growth and Fe(III) reduction by S. alga.Although not obligatory, nutrient coupling between these two organisms illustrates that fermentative (A. veronii-type) organisms can convert organic compounds such as citrate to those used as substrates by dissimilatory Fe(III) reducers, including S. alga. This synergism broadens the range of substrates available for iron reduction, stimulates the extent and rate of organic electron donor degradation (and that of iron reduction) and enhances the growth of each participant. Received: 11 December 1995 / Accepted: 19 June 1996     

Influence of citrate on the diacetyl and acetoin production by fully grown cells ofStreptococcus lactis subsp.diacetylactis

The effects of citrate on diacetyl and acetoin level by fully grown cells ofStreptococcus lactis subsp.diacetylactis CNRZ 124 were studied. In the absence of citrate, diacetyl synthase as well as acetolactate synthase and acetoin and diacetyl reductases exhibited a basal activity confirming their constitutive nature. However, when initial citrate concentration ranged from 8.8 to 59 mM, the enzyme levels increased in the same way, indicating no saturation rate of citrate metabolism. These results were reflected by a similar enhancement in acetoin and diacetyl production. When citrate was added in fed-batch conditions, its utilization by the fully grown cells led to a twofold increase in diacetyl yield over batch conditions.     

Fermentation of citrate by Lactobacillus plantarum in the presence of a yeast under acid conditions

Summary At pH 3.6, Lactobacillus plantarum is unable to grow on citrate or to ferment it in the absence of another carbon source such as glucose. In a defined medium containing glucose and citrate, with a higher concentration of the former than the latter, as in many fermented alcoholic beverages, L. plantarum will first ferment the sugar. The production of lactate from glucose degradation increases the acidity of the medium and inhibits the fermentation of citrate. In co-culture with Saccharomyces cerevisiae, part of the glucose is fermented by the yeast, partly avoiding the pH drop and the inhibition of citrate fermentation by L. plantarum. Fermentation was still possible at pH values around 3.0. Offprint requests to: C. Kennes     

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae. Received: 20 November 1996 / Accepted: 25 February 1997     

Characterization of a citrate-negative mutant ofLeuconostoc mesenteroides subsp.mesenteroides: metabolic and plasmidic properties

Summary Comparison of the parental strain of the Leuconostoc mesenteroides subsp. mesenteroides (19D) and its citrate-negative mutant, which has lost a 22-kb plasmid, has confirmed the energetic role of citrate. Fermentation balance analysis showed that citrate led to a change in heterolactic fermentation from glucose. High levels of enzyme activity in both mutant and parental strains were found for NADH oxidase, lactate dehydrogenase, acetate kinase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, although NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase were partly repressed by citrate. All these enzymes studied were not plasmid linked. In the parental strain, citrate lyase was induced by citrate. No citrate lyase activity was found in the citrate-negative mutant grown in presence of citrate, but this does not provide evidence that citrate lyase is linked to the 22-kb plasmid. Offprint requests to: C. Diviès     

The three tricarboxylate synthase activities of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and increase of <Emphasis Type="SmallCaps">l</Emphasis>-lysine synthesis

Corynebacterium glutamicum owns a citrate synthase and two methylcitrate synthases. Characterization of the isolated enzymes showed that the two methylcitrate synthases have comparable catalytic efficiency, k _cat/K _m, as the citrate synthase with acetyl-CoA as substrate, although these enzymes are only synthesized during growth on propionate-containing media. Thus, the methylcitrate synthases have a relaxed substrate specifity, as also demonstrated by their activity with butyryl-CoA, whereas the citrate synthase does not accept acyl donors other than acetyl-CoA. A double mutant deleted of the citrate synthase gene gltA and one of the methylcitrate synthase genes, prpC1, was made unable to grow on glucose. From this mutant, a collection of suppressor mutants could be isolated which were demonstrated to have regained citrate synthase activity due to the relaxed specificity of the methylcitrate synthase PrpC2. Molecular characterization of these mutants showed that the regulator PrpR (Cg0800) located downstream of prpC1 is mutated with mutations likely to effect the secondary structure of the regulator, thus, resulting in expression of prpC2. This expression results in a citrate synthase activity, which is lower than that due to gltA in the original strain and results in increased l-lysine accumulation.     

Flurorcitrate resistant tricarboxylate transport mutants of Salmonella typhimurium

Summary Spontaneous and Tn10 induced fluorocitrate resistant mutants were isolated and characterized. These mutants were unable to grow on either cis-aconitate or DL-isocitrate but were still able to grow slowly on sodium citrate and normally on potassium or potassium-plus-sodium citrate. These mutants were defective in both citrate transport and citrate binding to periplasmic proteins. Tn10 insertion mutants were unable to produce immunologically detectable amounts of the citrate inducible periplasmic C protein previously shown to bind tricarboxylates.Using a series of tct::Tn10 directed Hfrs the tct locus was accurately positioned at 59 units between srlA and pheA, but was not cotransducible with either gene. In the absence of P22 mediated cotransduction with 16 adjacent chromosomal markers the srlA and tct loci were bridged by using a series of tct flanking Tn10 insertions, and by newly isolated and characterized nalB mutants. In addition the hyd and recA loci were located establishing the gene order in this region of the chromosome as: pheA tct nalB recA srlA hyd cys. Nitrosoguanidine derived tricarboxylate mutations (Imai 1975) were also mapped within the tct locus.     

Origin of end-products from the co-metabolism of glucose and citrate by Leuconostoc mesenteroides subsp. cremoris

Summary The addition of citrate to glucose broth led to an increase in specific growth rate and glucose catabolism, but a decrease in molar growth yield from glucose, in Leuconostoc mesenteroides subsp. cremoris. Acetate and formate were produced during the stationary phase of growth. According to the fermentation balance, part of the acetate and lactate came from the pyruvate of citrate metabolism. L. mesenteroides subsp. cremoris incorporated radioactive metabolites from [1,5-¹⁴C] citrate into cell material, primarily into lipids. [U-¹⁴C] Glucose was not incorporated into cell material.     

Targeted expression of SbMATE in the root distal transition zone is responsible for sorghum aluminum resistance

Aluminum (Al) toxicity is one of the major limiting factors for crop production on acid soils that comprise significant portions of the world's lands. Aluminum resistance in the cereal crop Sorghum bicolor is mainly achieved by Al‐activated root apical citrate exudation, which is mediated by the plasma membrane localized citrate efflux transporter encoded by SbMATE. Here we precisely localize tissue‐ and cell‐specific Al toxicity responses as well as SbMATE gene and protein expression in root tips of an Al‐resistant near‐isogenic line (NIL). We found that Al induced the greatest cell damage and generation of reactive oxygen species specifically in the root distal transition zone (DTZ), a region 1–3 mm behind the root tip where transition from cell division to cell elongation occurs. These findings indicate that the root DTZ is the primary region of root Al stress. Furthermore, Al‐induced SbMATE gene and protein expression were specifically localized to the epidermal and outer cortical cell layers of the DTZ in the Al‐resistant NIL, and the process was precisely coincident with the time course of Al induction of SbMATE expression and the onset of the recovery of roots from Al‐induced damage. These findings show that SbMATE gene and protein expression are induced when and where the root cells experience the greatest Al stress. Hence, Al‐resistant sorghum plants have evolved an effective strategy to precisely localize root citrate exudation to the specific site of greatest Al‐induced root damage, which minimizes plant carbon loss while maximizing protection of the root cells most susceptible to Al damage.     

Effects of altered citrate synthase and isocitrate dehydrogenase expression on internal citrate concentrations and citrate efflux from tobacco (Nicotiana tabacum L.) roots

To assess the effectiveness of manipulating citrate metabolism with the aim of increasing citrate efflux from roots, we generated transgenic tobacco (Nicotiana tabacum L.) lines that either overexpressed mitochondrial citrate synthase (EC 4.1.3.7) activity or had reduced activity of cytosolic isocitrate dehydrogenase (EC 1.1.1.42). Despite increases in citrate synthase activities in transgenic lines of up to 5-fold, neither internal citrate concentrations nor citrate efflux were increased compared to controls suggesting that, in tobacco, citrate synthase activity does not directly determine citrate accumulation and efflux. Consistent with a lack of effect on citrate efflux, the increase in citrate synthase activity did not enhance the aluminium resistance of the transgenic lines. Preliminary data collected on two transgenic lines with cytosolic isocitrate dehydrogenase activities reduced to one-tenth and one third of the control for shoot and root tissues respectively, showed that while these changes in activities were associated with a 1.5-fold increase in internal citrate concentrations of both types of tissue, citrate efflux from roots was not increased. Further work is needed to establish whether the increase in internal citrate concentration is associated with enhanced aluminium resistance of these lines. We conclude that in tobacco internal citrate concentrations and citrate efflux are largely insensitive to large changes in either mitochondrial citrate synthase or cytosolic isocitrate dehydrogenase activities and suggest that other factors, such as transport out of the roots, control citrate efflux.