Divergent MLS1 Promoters Lie on a Fitness Plateau for Gene Expression.

Qualitative patterns of gene activation and repression are often conserved despite an abundance of quantitative variation in expression levels within and between species. A major challenge to interpreting patterns of expression divergence is knowing which changes in gene expression affect fitness. To characterize the fitness effects of gene expression divergence, we placed orthologous promoters from eight yeast species upstream of malate synthase (MLS1) in Saccharomyces cerevisiae As expected, we found these promoters varied in their expression level under activated and repressed conditions as well as in their dynamic response following loss of glucose repression. Despite these differences, only a single promoter driving near basal levels of expression caused a detectable loss of fitness. We conclude that the MLS1 promoter lies on a fitness plateau whereby even large changes in gene expression can be tolerated without a substantial loss of fitness.

The genome of the Antarctic polyextremophile Nesterenkonia sp. AN1 reveals adaptive strategies for survival under multiple stress conditions.

Nesterenkonia sp. AN1 is a polyextremophile isolated from Antarctic desert soil. Genomic analyses and genome comparisons with three mesophilic Nesterenkonia strains indicated that the unique genome fraction of Nesterenkonia sp. AN1 contains adaptive features implicated in the response to cold stress including modulation of membrane fluidity as well as response to cold-associated osmotic and oxidative stress. The core genome also encodes a number of putative cold stress response proteins. RNA-Seq-based transcriptome analyses of Nesterenkonia sp. AN1 grown at 5ºC and 21°C showed that there was significant induction of transcripts that code for antioxidants at 5ºC, demonstrated by the upregulation of sodA, bcp and bpoA2. There was also overexpression of universal stress protein genes related to uspA, along with genes encoding other characterized cold stress features. Genes encoding the two key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (AceB) were induced at 5ºC, suggesting possible adaptation strategies for energy metabolism in cold habitats. These genomic features may contribute to the survival of Nesterenkonia sp. AN1 in arid Antarctic soils.

Effect of unsaturated fatty acids on the biosynthesis of glucose-repressed enzymes in yeast.

In anaerobically glucose-grown yeast isocitrate lyase (EC 4.1.3.1.), and malate dehydrogenase (EC 1.1.1.37.) are repressed by glucose. 24 h cultures still contain 0.3--0.4% glucose in the medium, which is enough to completely repress these activities. Aeration of these cells, in buffer containing acetate, initiates the formation of the three enzymes. Within 16 h, the specific activities of these enzymes increase about 140, 120 and 70-fold, respectively. Glucose-6-phosphate dehydrogenase activity was not altered. When the yeast was grown anaerobically, but with a supplement of an unsaturated fatty acid in the medium, synthesis of the three enzymes was much faster and the specific activities after 16 h of derepression were considerably higher. A relationship exists between the number of double bonds in the unsaturated fatty acid molecule and its capability to stimulate enzyme synthesis: linolenic acid is more effective than linoleic acid, which, in turn, is much more effective than oleic acid. Increasing periods of aeration with glucose of anaerobically grown cells prior to derepression results in an increasing stimulation of enzyme synthesis on subsequent derepression. Anaerobic incubation of yeast in the presence of an unsaturated fatty acid in advance to derepression also increased the velocity of enzyme formation. It is suggested that during the aeration period with glucose and during anaerobic incubation with an unsaturated fatty acid a more active protein synthesizing apparatus was formed.

Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae.

We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was abolished. This indicated that derepression of UPR-MS-2 -mediated gene expression was dependent on Snf1p, as is the case of genes encoding isocitrate lyase and gluconeogenic enzymes in S. cerevisiae.

Induction of glyoxylate cycle enzymes in rat liver upon food starvation.

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of food-starved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K(m)(isocitrate) was 68 microM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K(m)(acetyl-CoA) of 0.2 microM, a K(m)(glyoxylate) of 3 mM and a pH optimum of 7.6.

[Induction of glyoxylate cycle enzymes in various tissues from starving rats]. / Induktsiia fermentov glioksilatnogo tsikla v razlichnykh tkaniakh golodaiushchikh krys.

The induction of glyoxylate cycle enzyme activities was revealed in the liver and other organs of starving rats. A five day deprivation of food was followed by the appearance of isocitrate lyase (ICL) and malate synthase activities and the increase of malate dehydrogenase (MDH) and citrate synthase activities. The induction of MDH was associated with the appearance of its new isoform with Rf 0.52. ICL activity was revealed in the liver, blood, pancreas, kidney, lungs, heart, and skeletal muscles of starving rats, reaching a peak on day 5 of food deprivation. No significant changes of blood glucose level in starving rats were revealed until day 9. A homogeneous ICL preparation with a specific activity of 12.4 IU per mg protein was obtained as the results of five-stage purification procedure.

Functional characterization of the glxR deletion mutant of Corynebacterium glutamicum ATCC 13032: involvement of GlxR in acetate metabolism and carbon catabolite repression.

Recently, a cyclic AMP receptor protein homologue, GlxR, was reported to bind to the upstream regions of several genes involved in the regulation of diverse physiological processes in Corynebacterium glutamicum. However, the function of GlxR has not yet been explored in C. glutamicum in vivo using a glxR deletion mutant. Therefore, this study examines the role of GlxR as a repressor in glyoxylate bypass and carbon catabolite repression (CCR) using a deletion mutant. The disruption of glxR resulted in a severe growth defect, but growth was restored by complementation with the glxR and crp genes from C. glutamicum and Streptomyces coelicolor, respectively. The production of isocitrate lyase (ICL) and malate synthase (MS) was significantly increased in the glxR mutant. The specific activities of both enzymes were increased in the glxR mutant, regardless of the carbon source. In accordance, the promoter activities of ICL and MS using lacZ fusion were derepressed in the glxR mutant. In addition, the glxR mutant exhibited derepression of the gluA gene for glutamate uptake in the presence of glucose, thereby relieving CCR by glucose. These results indicate that GlxR plays an important role in CCR as well as in acetate metabolism.

Anaerobic induction of isocitrate lyase and malate synthase in submerged rice seedlings indicates the important metabolic role of the glyoxylate cycle.

The glyoxylate cycle is a modified form of the tricarboxylic acid cycle that converts C2 compounds into C4 dicarboxylic acids at plant developmental stages. By studying submerged rice seedlings, we revealed the activation of the glyoxylate cycle by identifying the increased transcripts of mRNAs of the genes of isocitrate lyase (ICL) and malate synthase (MS), two characteristic enzymes of the glyoxylate cycle. Northern blot analysis showed that ICL and MS were activated in the prolonged anaerobic environment. The activity assay of pyruvate decarboxylase and ICL in the submerged seedlings indicated an 8.8-fold and 3.5-fold increase over that in the unsubmerged seedlings, respectively. The activity assay of acetyl-coenzyme A synthetase in the submerged seedlings indicated a 3-fold increase over that in the unsubmerged seedlings, which is important for initiating acetate metabolism. Consequently, we concluded that the glyoxylate cycle was involved in acetate metabolism under anaerobic conditions.

Rates of de novo synthesis of malate synthase and albumins during the very early phase of germination.

Malate synthase is synthesized de novo in the very phase of germination. Its molecular and immunological properties do not differ from those of malate synthase from fully developed cotyledons. Radioactive leucine was administered to dry seeds of cucumber, and its incorporation into proteins of cotyledons was examined after 2 days of germination. The specific radioactivity of malate synthase, purified by immunoprecipitation and electrophoresis on polyacrylamide gel, was only 1/20 the average value of the total albumin fraction. The minimal incorporation documented by the comparatively low specific activity of isolated malate synthase is discussed in relation to the large pool of malate synthase already present in dry seeds.

Studies supporting the concept of glyoxyperoxisomes as intermediary organelles in transformation of glyoxysomes into peroxisomes.

Intermediary forms of microbodies, glyoxyperoxisomes, are most likely synthesized at the stage of replacement of glyoxysomes by peroxisomes during the greening of fatstoring cells of cucumber cotyledons. This hypothesis is supported by three lines of results: 1. The levels of both glycollate oxidase and malate synthase are increased in the microbody fractions upon illumination of cotyledons. 2. Both peroxisomal and glyoxysomal marker enzymes are localized in organelles which exhibit the same buoyant densities. 3. De novo synthesis of malate synthase takes place even when irradiation already leads to a drastic increase of glycollate oxidase. The amount of malate synthase synthesized is almost identical in the light-induced system and in the dark control.

Stimulation of isocitrate lyase biosynthesis by hydroxylamine and hydrazine.

Recently it has been demonstrated that hydroxylamine is an activator of triglyceride catabolism. We have studied the effect of hydroxylamine on isocitrate lyase activity and lipid catabolism and have noted a stimulation of isocitrate lyase biosynthesis by 5 mM hydroxylamine. The specificity of this effect was tested with a number of representative enzymes of other metabolic pathways. In an attempt to study the possible mechanism of action of hydroxylamine we have also tested the effects of two substances that are structural or functional analogues of hydroxylamine, namely, ethanolamine and hydrazine, both on the enzyme level in plant cultures and on the activity of enzyme preparations. From our data we may conclude that "de nove" biosynthesis of isocitrate lyase depends on the reaction of hydroxylamine or hydrazine with glyoxylate to give the corresponding oxime and hydrazone. The removal of glyoxylate from the biological equilibrium in this way could cause extra formation of isocitrate lyase.

Regulation of glyoxysomal enzymes during germination of cucumber. Temporal changes in translatable mRNAs for isocitrate lyase and malate synthase.

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.

[Regulation of glyoxylate cycle enzymes in Saccharomycopsis lipolytica. I. Effect of the carbon source on isocitrate lyase and malate synthase activity]. / Untersuchungen zur Regulation der Enzyme des Glyoxylatzyklus bei Saccharomycopsis lipolytica. I. Einfluss der C-Quelle auf die Aktivität von Isocitratlyase und Malatsynthase.

Comparative studies on the activities of isocitrate lyase (ICL) and malate synthase (MS) were carried out with Saccharomycopsis lipolytica incubating the yeast on media with different carbon sources. When cells were incubated in minimal medium with glucose, the activities of both enzymes were very low. In contrast, in minimal medium with acetate enhanced enzyme activities could be demonstrated. It is probably that the synthesis of ICL is repressed in presence of glucose. Furthermore the activity of ICL was inhibited by tricarboxylic acid cycle intermediates like succinic acid and oxalacetic acid. It was concluded that the syntheses of enzymes are derepressed. When cells of Sm. lipolytica were incubated in minimal medium with acetate, a high enzyme activity is evident. Synthesis of ICL on acetate was inhibited by cycloheximide and actinomycin D. The results were discussed comparing them with data obtained from other organisms.

Expression of a single gene encoding microbody NAD-malate dehydrogenase during glyoxysome and peroxisome development in cucumber.

A full-length cDNA clone encoding microbody NAD(+)-dependent malate dehydrogenase (MDH) of cucumber has been isolated. The deduced amino acid sequence is 97% identical to glyoxysomal MDH (gMDH) of watermelon, including the amino terminal putative transit peptide. The cucumber genome contains only a single copy of this gene. Expression of this mdh gene increases dramatically in cotyledons during the few days immediately following seed imbibition, in parallel with genes encoding isocitrate lyase (ICL) and malate synthase (MS), two glyoxylate cycle enzymes. The level of MDH, ICL and MS mRNAs then declines, but then MDH mRNA increases again together with that of peroxisomal NAD(+)-dependent hydroxypyruvate reductase (HPR). The mdh gene is also expressed during cotyledon senescence, together with hpr, icl and ms genes. These results indicate that a single gene encodes MDH which functions in both glyoxysomes and peroxisomes. In contrast to icl and ms genes, expression of the mdh gene is not activated by incubating detached green cotyledons in the dark, nor is it affected by exogenous sucrose in the incubation medium. The function of this microbody MDH and the regulation of its synthesis are discussed.

The glcB locus of Rhizobium leguminosarum VF39 encodes an arabinose-inducible malate synthase.

In the course of a study conducted to isolate genes upregulated by plant cell wall sugars, we identified an arabinose-inducible locus from a transcriptional fusion library of Rhizobium leguminosarum VF39, carrying random insertions of the lacZ transposon Tn5B22. Sequence analysis of the locus disrupted by the transposon revealed a high similarity to uncharacterized malate synthase G genes from Sinorhizobium meliloti, Agrobacterium tumefaciens, and Mesorhizobium loti. This enzyme catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate and CoA and is thought to be a component of the glyoxylate cycle, which allows microorganisms to grow on two carbon compounds. Enzyme assays showed that a functional malate synthase is encoded in the glcB gene of R. leguminosarum and that its expression is induced by arabinose, glycolate, and glyoxylate. An Escherichia coli aceB glcB mutant, complemented with the R. leguminosarum PCR-amplified gene, recovered malate synthase activity. A very similar genome organization of the loci containing malate synthase and flanking genes was observed in R. leguminosarum, S. meliloti, and A. tumefaciens. Pea plants inoculated with the glcB mutant or the wild-type strain showed no significant differences in nitrogen fixation. This is the first report regarding the characterization of a mutant in one of the glyoxylate cycle enzymes in the rhizobia.

Purification and characterization of recombinant malate synthase enzymes from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585.

Malate synthases (MS) from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585 were cloned by polymerase chain reaction into a glutathione S-transferase (GST) fusion expression vector and heterologously expressed in Escherichia coli. The fusion GST-MS construct improved the soluble expression of MS by approximately 10-fold compared to the soluble expression of nonfusion MS. With the significant improvement in levels of soluble MS, purification and subsequent cleavage of recombinant MS from GST were facilitated in this study. Using purified enzymes, optimized parameters, which achieved maximal specific activity, were established in the enzymatic assay for streptomycete MS. The average purified specific activities of S. coelicolor and S. clavuligerus MS were 26199 and 11821 nmol/mg min, respectively. Furthermore, enzymatic analysis revealed that the two streptomycete MS displayed a similar Km value for acetyl-CoA, but S. coelicolor MS had a Km value for glyoxylate that is approximately sixfold higher than S. clavuligerus MS.

Molecular characterization of Escherichia coli malate synthase G. Differentiation with the malate synthase A isoenzyme.

Two genes encoding the enzymes malate synthase G and glycolate oxidase, have been linked to locus glc (64.5 min), responsible for glycolate utilization in Escherichia coli. The gene encoding malate synthase G, for which we propose the notation glcB, has been cloned, sequenced and found to correspond to a 2262-nucleotide open-reading frame, which can encode a 723-amino-acid polypeptide, clearly different from the isoenzyme malate synthase A, which has 533 amino acids. Northern-blot experiments indicate that glcB was expressed as an apparently monocistronic transcript, inducible by glycolate. Malate synthase G was purified to near homogeneity. The molecular mass determined by gel filtration yielded a value of 82 kDa for the purified enzyme and the same value as for the crude extract enzyme, indicating a monomeric structure. Despite the lower sequence similarity between malate synthase G and the other reported malate synthases, three out of nine consensus boxes defined in most of these enzymes are conserved in addition to a cysteine residue that has been reported to be important for the catalytic mechanisms.

The glyoxylate cycle is required for fungal virulence.

Candida albicans, a normal component of the mammalian gastrointestinal flora, is responsible for most fungal infections in immunosuppressed patients. Candida is normally phagocytosed by macrophages and neutrophils, which secrete cytokines and induce hyphal development in this fungus. Neutropenic patients, deficient in these immune cells, are particularly susceptible to systemic candidiasis. Here we use genome-wide expression profiles of the related yeast Saccharomyces cerevisiae to obtain a signature of the events that take place in the fungus on ingestion by a mammalian macrophage. Live S. cerevisiae cells isolated from the phagolysosome are induced for genes of the glyoxylate cycle, a metabolic pathway that permits the use of two-carbon compounds as carbon sources. In C. albicans, phagocytosis also upregulates the principal enzymes of the glyoxylate cycle, isocitrate lyase (ICL1) and malate synthase (MLS1). Candida albicans mutants lacking ICL1 are markedly less virulent in mice than the wild type. These findings in fungi, in conjunction with reports that isocitrate lyase is both upregulated and required for the virulence of Mycobacterium tuberculosis, demonstrate the wide-ranging significance of the glyoxylate cycle in microbial pathogenesis.

Relative expression of the products of glyoxylate bypass operon: contributions of transcription and translation.

Although the genes of the aceBAK operon are expressed from the same promoter, the relative cellular levels of their products are approximately 0.3:1:0.003. Gene and operon fusions with lacZ were constructed to characterize this differential expression. The upshift in expression between aceB and aceA resulted from differences in translational efficiency. In contrast, inefficient translation and premature transcriptional termination contributed to the downshift in expression between aceA and aceK. Premature transcriptional termination occurred within aceK and appears to result from inefficient translation. Deletion of repetitive extragenic palindromic elements between aceA and aceK had little effect on the relative expression of these genes. Rather, the sequences responsible for inefficient expression of aceK lie within the aceK ribosome binding site.