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Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus. We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt. IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus. Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.

Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria ( Euryarchaeota ). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica ) and β-methylmalyl-CoA lyase (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave β-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a β-methylmalyl-CoA lyase reaction in vivo . The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides , were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3 S )-malyl-CoA lyase operating in the ethylmalonyl-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3 S )-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3 S )-malyl-CoA but does not use β-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as “Claisen condensation” enzymes so far. Mcl1 and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction.

In Corynebacterium glutamicum , the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum , showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG 4-6 T/C or AC 4-5 A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum .

A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum , was analyzed further. In the clone, an open reading frame designated glxR , consisting of 681 nucleotides and encoding a 24,957-Da protein, was found. The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers. The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins. The aceB -repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP. In glucose medium, the intracellular cAMP concentration of C. glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase. The expression of glxR was not affected by the carbon source. Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP. These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.

The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB. The gene glcC may encode the glc regulator protein. Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities. The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins. The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of this enzyme. No effect on glycolate metabolism was detected by insertional mutation in glcG. Northern (RNA) blot experiments showed constitutive expression of glcC but induced expression for the structural genes and provided no evidence for a single polycistronic transcript.

The aceBAK operon was partially induced by a multicopy plasmid which carried the promoter region of the gene which encodes its repressor, iclR. Gel shift and DNase I analyses demonstrated that IclR binds to its own promoter. Disruption of iclR increased the expression of an iclR::lacZ operon fusion. Although aceBAK and iclR are both regulated by IclR, aceBAK expression responds to the carbon source, while expression of iclR does not.

Acetone degradation by cell suspensions of Desulfococcus biacutus was CO2 dependent, indicating initiation by a carboxylation reaction, while degradation of 3-hydroxybutyrate was not CO2 dependent. Growth on 3-hydroxybutyrate resulted in acetate accumulation in the medium at a ratio of 1 mol of acetate per mol of substrate degraded. In acetone-grown cultures no coenzyme A (CoA) transferase or CoA ligase appeared to be involved in acetone metabolism, and no acetate accumulated in the medium, suggesting that the carboxylation of acetone and activation to acetoacetyl-CoA may occur without the formation of a free intermediate. Catabolism of 3-hydroxybutyrate occurred after activation by CoA transfer from acetyl-CoA, followed by oxidation to acetoacetyl-CoA. In both acetone-grown cells and 3-hydroxybutyrate-grown cells, acetoacetyl-CoA was thioyltically cleaved to two acetyl-CoA residues and further metabolized through the carbon monoxide dehydrogenase pathway. Comparison of the growth yields on acetone and 3-hydroxybutyrate suggested an additional energy requirement in the catabolism of acetone. This is postulated to be the carboxylation reaction (delta G(o)' for the carboxylation of acetone to acetoacetate, +17.1 kJ.mol-1). At the intracellular acyl-CoA concentrations measured, the net free energy change of acetone carboxylation and catabolism to two acetyl-CoA residues would be close to 0 kJ.mol of acetone-1, if one mol of ATP was invested. In the absence of an energy-utilizing step in this catabolic pathway, the predicted intracellular acetoacetyl-CoA concentration would be 10(13) times lower than that measured. Thus, acetone catabolism to two acetyl-CoA residues must be accompanied by the utilization of teh energetic equivalent of (at lease) one ATP molecule. Measurement of enzyme activities suggested that assimilation of acetyl-CoA occurred through a modified citric acid cycle in which isocitrate was cleaved to succinate and glyoxylate. Malate synthase, condensing glyoxylate and acetyl-CoA, acted as an anaplerotic enzyme. Carboxylation of pyruvate of phosphoenolpyruvate could not be detected.

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.

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C 1 and C 2 compounds, showing that both modes of growth have this pathway in common. However, growth on C 2 compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/β-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C 2 compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and 13 C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/β-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C 2 compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H 4 F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C 2 compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.

For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase. The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme's activity and thus forcing isocitrate through the bypass. This phosphorylation cycle is catalyzed by a bifunctional enzyme, IDH kinase/phosphatase, which is encoded by aceK. We have employed random mutagenesis to isolate novel alleles of aceK. These alleles were detected by the loss of ability to complement an aceK null mutation. The products of one class of these alleles retain IDH kinase activity but have suffered reductions in IDH phosphatase activity by factors of 200 to 400. Selective loss of the phosphatase activity also appears to have occurred in vivo, since cells expressing these alleles exhibit phenotypes which are reminiscent of strains lacking IDH; these strains are auxotrophic for glutamate. Assays of cell-free extracts confirmed that this phenotype resulted from nearly quantitative phosphorylation of IDH. The availability of these novel alleles of aceK allowed us to assess the significance of the precise control which is a characteristic of the IDH phosphorylation cycle in vivo. The fractional phosphorylation of IDH was varied by controlled expression of one of the mutant alleles, aceK3, in a wild-type strain. Reduction of IDH activity to 50% of the wild-type level did not adversely affect growth on acetate. However, further reductions inhibited growth, and growth arrest occurred when the IDH activity fell to 15% of the wild-type level. Thus, although wild-type cells maintain a precise effective IDH activity during growth on acetate, this precision is not critical.

In Escherichia coli, expression of the glyoxylate bypass operon appears to be controlled, in part, by the product of iclR+. Mutations in iclR have been found to yield constitutive expression of this operon, suggesting that iclR+ encodes a repressor protein. We have cloned iclR+ by taking advantage of its tight genetic linkage with the glyoxylate bypass operon. The clone complemented a mutant allele of iclR in trans, restoring an inducible phenotype for this operon. Deletion analysis identified a region of ca. 900 base pairs that was necessary and sufficient for complementation. The nucleotide sequence of the insert was then determined. Translation of this sequence revealed an open reading frame capable of encoding a protein with Mr 29,741 preceded by a potential Shine-Dalgarno ribosome-binding site. The deduced amino acid sequence includes a region at the amino terminus that may form a helix-turn-helix motif, a structure found in many DNA-binding domains.

Cells of Escherichia coli increase greatly the synthesis of a small cytoplasmic protein as soon as the cell growth rate falls below the maximal growth rate supported by the medium, regardless of the condition inhibiting growth. The gene, designated uspA (universal stress protein A), encoding this protein has been cloned and mapped, and its nucleotide sequence has been determined (T. Nyström and F.C. Neidhardt, Mol. Microbiol. 6:3187-3198, 1992). We now report the isolation of an E. coli mutant defective in UspA synthesis because of insertional inactivation of the corresponding gene. Analysis of such a mutant demonstrated that it grows at a rate indistinguishable from that of the isogenic parent but lags significantly when diluted into fresh medium, regardless of the carbon source included. In addition, the mutant exhibits a diauxic type of growth when grown on certain single substrates, such as glucose and gluconate. This growth phenotype was found to be the result of abnormal metabolism of the carbon source (e.g., glucose) accompanied by excretion into the medium of acetate. The diauxic type of growth may be attributed to the failure of cells to form acetyl coenzyme A synthetase and to form isocitrate lyase and malate synthase of the glyoxalate bypass, needed for the assimilation of the produced acetate, until glucose or gluconate has been completely exhausted. The uspA mutant appears to dissimilate glucose at an elevated rate that is not commensurate with its biosynthetic processes. These results suggest that the role of protein UspA may be to modulate and reorganize the flow of carbon in the central metabolic pathways of E. coli during growth arrest.

Expression of beta-galactosidase in transcriptional fusions with the pps gene (encoding phosphoenolpyruvate [PEP] synthase), the aceBAK operon (encoding malate synthase, isocitrate lyase, and isocitrate dehydrogenase kinase, respectively), and the phs operon (encoding either thiosulfate reductase or a regulatory protein controlling its expression) was studied in Salmonella typhimurium. beta-Galactosidase synthesis in these strains was repressible either by growth in the presence of glucose or by the presence of a fruR mutation, which resulted in the constitutive expression of the fructose (fru) regulon. Five enzymes of gluconeogenesis (PEP synthase, PEP carboxykinase, isocitrate lyase, malate synthase, and fructose-1,6-diphosphatase) were shown to be repressed either by growth in the presence of glucose or the fruR mutation, while the glycolytic enzymes, enzyme I and enzymes II of the phosphotransferase system as well as phosphofructokinase, were induced either by growth in the presence of glucose or the fruR mutation. Overexpression of the cloned fru regulon genes (not including fruR) resulted in parallel repression of representative gluconeogenic, Krebs cycle, and glyoxylate shunt enzymes. Studies with temperature-sensitive mutants of S. typhimurium which synthesized heat-labile IIIFru proteins provided evidence that this protein plays a role in the regulation of gluconeogenic substrate utilization. Other mutant analyses revealed a complex relationship between fru gene expression and the expression of genes encoding gluconeogenic enzymes. Taken together, the results suggest that a number of genes encoding catabolic, biosynthetic, and amphibolic enzymes in enteric bacteria are transcriptionally regulated by a complex catabolite repression/activation mechanism which may involve enzyme IIIFru of the phosphotransferase system as one component of the regulatory system.

Malate synthases from a thermophilic Bacillus and Escherichia coli have been isolated in a high state of purity. Molecular weights of these two proteins determined in the native state and after denaturation in sodium dodecyl sulfate-mercaptoethanol show that the enzymes are monomeric. This conclusion is supported, for the thermophile enzyme, by the result of an electrophoretic analysis of that protein after treatment with dimethylsuberimidate and denaturation. The thermophilic Bacillus malate synthase is considerably more thermostable than its mesophilic counterparts from E. coli, Bacillus licheniformis, and Pseudomonas indigofera. It is, however, markedly labilized by an increase in the ionic strength of the medium brought about by the addition of 0.2 M potassium chloride or in pH above 9. Increased ionic strength has little effect on the thermostability of the mesophilic bacterial malate synthases. These observations provide strong support for the idea that monomeric proteins in thermophiles owe their unusual heat stability to the presence of salt bridges in their tertiary structure.

Mutants of Escherichia coli K-12 constitutive for the synthesis of the enzymes of fatty acid degradation (fadR) have elevated levels of the glyoxylate shunt enzymes, isocitrate lyase and malate synthase. A temperature-sensitive fadR strain has high levels of glyoxylate shunt enzymes when grown at elevated temperatures but has low, inducible levels of glyoxylate shunt enzymes when grown at low temperatures. The increased activity of glyoxylate shunt enzymes did not appear to be due to the degradation of intracellular fatty acids in fadR strains or differences in allosteric effectors in fadR versus fadR+ strains. These studies suggest that the fadR gene product may be involved in the regulation of the glyoxylate operon.

Isocitrate lyase and malate synthase, the two unique enzymes of the glyoxylate cycle, were detected in crude extracts of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Y. pestis, unlike Escherichia coli and the other yersiniae tested, yielded two forms of isocitrate lyase during growth on acetate. These forms differed in electrophoretic mobility and temperature optima. One form (A) was present during growth on acetate, but was absent during growth on alternate carbon sources such as glucose. The second form (B) was not constitutive, but was found during growth on acetate, glucose, xylose, or other complex carbon sources. Itaconate, a succinate analog which inhibited both forms of isocitrate lyase in crude extracts, did not affect the growth of Y. pestis under conditions where little isocitrate lyase activity was detected. This inhibitor, however, retarded the growth of Y. pestis under conditions where acetate was provided as the primary carbon and energy source as well as under all conditions in which either form of isocitrate lyase was evident. This suggests that the B form may play an important role in the growth of this bacterium under conditions where a requirement for the classical anaplerotic sequence involving this enzyme is not apparent.

Mutants of Candida lipolytica that were unable to grow on acetate but able to utilize succinate or glycerol as a sole carbon source were isolated. Amongst the mutants isolated, one strain (Icl-) was specifically deficient in isocitrate lyase activity, whereas another strain (Acos-) was deficient in acetyl coenzyme A synthetase activity. Since the Icl- mutant could not grow either on n-alkane or its derivatives, such as fatty acid and long-chain dicarboxylic acid, any anaplerotic route other than the glyoxylate pathway was inconceivable as far as growth on these carbon sources was concerned. Acetyl coenzyme A is most likely a metabolic inducer of isocitrate lyase and malate synthase, because the Acos- mutant was characterized by the least susceptibility to induction of these enzymes by acetate. The structural gene for isocitrate lyase was most probably impaired in the Icl- mutant, since revertants (Icl-) produced thermolabile isocitrate lyase. The production of isocitrate from n-alkane by the revertants was enhanced in comparison with the parental strain.

The isocitrate lyase from a thermophilic Bacillus is activated about threefold by a variety of salts. Such strong stimulation of activity is not seen with isocitrate lyase from the mesophiles, Bacillus licheniformis, Bacillus megaterium, Escherichia coli , and Aspergillus nidulans . The salt activation is markedly pH-dependent. At pH values above 8.6, salt (KCl) indeed inhibits the enzyme activity. Potassium chloride also causes a significant shift of the pH optimum of the enzyme towards the acid side. As the temperature of the enzyme reaction is raised, activation becomes progressively weaker. Potassium chloride also affords considerable protection against enzyme denaturation at 55 C. The activation and the stabilization, however, appear to be independent effects. Of six other enzymes in the thermophile that were examined, isocitrate dehydrogenase was equally strongly activated by KCl and malate synthase was less strongly, but significantly, activated; citrate synthase, malate dehydrogenase, glutamate dehydrogenase, and lactate dehydrogenase were unaffected or slightly inhibited by KCl. The property of being strongly activated by salt appears to be a peculiar characteristic of the thermophile isocitrate lyase and possibly evolved concomitantly with its thermostability.

Wegener, Warner S. (Albert Einstein Medical Center, Philadelphia, Pa.), Henry C. Reeves, and Samuel J. Ajl . Heterogeneity of the glyoxylate-condensing enzymes. J. Bacteriol. 90: 594–598. 1965.—Evidence is presented that the enzymatic condensations of glyoxylate with acetyl-CoA (malate synthase), propionyl-CoA (α-hydroxyglutarate synthase), butyryl-CoA (β-ethylmalate synthase), and valeryl-CoA (β- n -propylmalate synthase) are catalyzed by different enzymes. The possibility that these activities resulted from a single enzyme possessing a broad fatty acid acyl-CoA substrate specificity was ruled out. The latter was suggested by the fact that cells grown on a number of short-chain fatty acids exhibited all the above activities. The conclusion that these reactions are catalyzed by different enzymes is based on the following considerations: (i) the enzymes can be differentially inactivated by heat; (ii) under various growth conditions, where all the condensing enzymes are present, their respective activities do not show a constant ratio, as would be expected if they were catalyzed by a single enzyme; and (iii) under appropriate growth conditions, one or more of these enzymes has been shown to be present to the exclusion of others.

The relative contributions of the dicarboxylic acid and the tricarboxylic acid cycles to the oxidative catabolism of glyoxylate in Escherichia coli K-12 were deduced by analysis of mutant strains that were blocked in the formation of glyoxylate carboligase and of malate synthase G (the “glycolate form” of malate synthase). Mutant strains unable to form malate synthase G were unimpaired in their ability to oxidize glyoxylate. Hence, the dicarboxylic acid cycle does not appear to play an essential role in this process. Organisms blocked in the synthesis of glyoxylate carboligase did not oxidize glyoxylate at a detectable rate, indicating that wild-type organisms convert glyoxylate to acetyl-coenzyme A and oxidize it via the tricarboxylic acid cycle. The foregoing evidence indicates that malate synthase G plays an anaplerotic role during growth with glycolate or acetate as the carbon source. The in vivo activity of malate synthase G was not detectable when the intracellular concentration of acetyl-coenzyme A was low, suggesting that this substrate or a closely related metabolite exerts a sensitive positive control over the enzyme. The synthesis of malate synthase G appears to be induced directly by glycolate which may be formed by a constitutive reduced nicotinamide adenine dinucleotide phosphate-dependent glyoxylate reductase in glyoxylate- or acetate-grown cells.

Reeves, Henry C. (Albert Einstein Medical Center, Philadelphia, Pa.) and Samuel J. Ajl . Function of malate synthetase and isocitritase in the growth of bacteria on two-carbon compounds. J. Bacteriol. 83: 597–601. 1962.— Escherichia coli , grown in a glucose-citrate medium under anaerobic conditions, is devoid of the enzymes malate synthetase and isocitritase. It has been demonstrated that the adaptation of such cells to aerobic growth in a medium containing acetate as the sole carbon source involves at least two separate events. The first appears to be an adaptation which permits the oxidative dissimilation of acetate and involves primarily the synthesis of condensing enzyme. The second, which also precedes all division, is the synthesis of isocitritase and malate synthetase. Finally, a definite correlation has been found to exist between the synthesis of isocitritase and malate synthetase and the lag period prior to cell proliferation.

The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing ( acu ) mutants of Neurospora crassa , both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed.

Howes, William V. (Washington State University, Pullman) and Bruce A. McFadden . Isocitrate lyase and malate synthase in Pseudomonas indigofera . II. Enzyme changes during the phase of adjustment and the early exponential phase. J. Bacteriol. 84: 1222–1227. 1962.—The differential rates of synthesis (DRS) of malate synthase and isocitrate lyase were investigated at 30 C during the phase of adjustment (lag) and the early exponential phase in Pseudomonas indigofera . Cells grown on ethanol-acetate-yeast extract or succinate-yeast extract were added to a medium containing ethanol or succinate, and the DRS of each enzyme was followed. A reproducible decline in both enzymatic activities occurred after inoculation when a lag in growth was observed. To define the DRS occurring after this decline, a new parameter, ε, was defined and calculated for linear segments of a DRS plot. Certain variations of ε during DRS suggested that a balance between induction (or derepression) and repression controls, in part at least, malate synthase and isocitrate lyase levels. Effects of chloramphenicol upon enzyme synthesis after the period of expected decline were studied. No degradation or synthesis of malate synthase occurred in the presence of chloramphenicol, although, after degradation, slight synthesis of isocitrate lyase occurred. Acetate, butyrate, ethanol, glyoxylate, and isocitrate did not stimulate production of either enzyme in nongrowing cultures of high cell density. In fact, some degradation of each enzyme occurred.

The metabolism of propane and propionate by a soil isolate (Brevibacteriumsp. strain JOB5) was investigated. The presence of isocitrate lyase in cells grown on isopropanol, acetate, or propane and the absence of this inducible enzyme inn-propanol- and propionate-grown cells suggested that propane is not metabolized via C-terminal oxidation. Methylmalonyl coenzyme A mutase and malate synthase are constitutive in this organism. The incorporation of14CO2into pyruvate accumulated during propionate utilization suggests that propionate is metabolized via the methyl-malonyl-succinate pathway. These results were further substantiated by radiorespirometric studies with propionate-1-14C, -2-14C, and -3-14Cas substrate. Propane -2-14Cwas shown, by unlabeled competitor experiments, to be oxidized to acetone; acetone and isopropanol are oxidized in this organism to acetol. Cleavage of acetol to acetate and CO2would yield the inducer for the isocitrate lyase present in propane-grown cells.

An inducible beta-oxidation system was demonstrated in a particulate fraction from Neurospora crassa. The activities of all individual beta-oxidation enzymes were enhanced in cells after a shift from a sucrose to an acetate medium. The induction was even more pronounced in transfer to a medium containing oleate as sole carbon and energy source. Since an acyl-coenzyme A (CoA) dehydrogenase was detected instead of acyl-CoA oxidase, the former enzyme seems to catalyze the first step of the beta-oxidation sequence in N. crassa. After isopycnic centrifugation in a linear sucrose gradient, the intracellular organelles housing the fatty acid degradation pathway cosedimented (1.21 g/cm3) with the glyoxylate bypass enzymes isocitrate lyase and malate synthase and were clearly resolved from both mitochondrial marker enzymes (1.19 g/cm3) and catalase (1.26 g/cm3). On the basis of biochemical as well as morphological properties, these particles from N. crassa have recently been designated as glyoxysome-like particles (G. Wanner and T. Theimer, Ann. N.Y. Acad. Sci. 386:269-284, 1982). The failure to detect catalase, urate oxidase, and acyl-CoA oxidase indicate that these glyoxysome-like microbodies in N. crassa lack peroxisomal function and thus are clearly different from the various microbodies reported so far to contain a beta-oxidation pathway.

meso-Tartrate inhibited the growth of non-meso-tartrate-utilizing strains of Salmonella typhimurium in peptone water media and mineral salts media with some, but not all, carbon sources. C-R intermediates of the tricarboxylic acid cycle or compounds readily converted to them and substrates metabolized independently of the C-6 part of the cycle spared bacteria from the inhibitory effects of meso-tartrate when added to cultures along with meso-tartrate. Experiments with cell-free extracts of non-meso-tartrate-utilizing strains from batch and continuous cultures showed that meso-tartrate was a competitive inhibitor of isocitrate dehydrogenase and isocitrate lyase activities and also inhibited citrate synthase and malate synthase activities. The synthesis of these enzymes was not inhibited by meso-tartrate. The isocitrate enzymes of meso-tartrate-utilizing strains of S. typhimurium were similarly inhibited by meso-tartrate, but inhibition of the growth of meso-tartrate-utilizing strains was demonstrable only in uninduced cultures in which the intracellular concentrations of meso-tartrate were high.

Growth of Salmonella typhimurium on acetate as a sole carbon source requires expression of the glyoxylate shunt; however, the genes for the glyoxylate shunt enzymes have not been previously identified in S. typhimurium. In this study, we isolated transposon insertions in the genes for the two unique enzymes of this pathway, aceA (isocitrate lyase) and aceB (malate synthase). The aceA and aceB genes were located at 89.5 min on the S. typhimurium genetic map. Genetic linkage to nearby loci indicated that the relative gene order is purDJ metA aceB aceA. Transposon insertions in aceB were polar on aceA, suggesting that the genes form an operon transcribed from aceB to aceA. Transcriptional regulation of the aceBA operon was studied by constructing mini-Mu d(lac Kan) operon fusions. Analysis of these fusions indicated that expression of the aceBA operon is regulated at the level of transcription; the aceBA genes were induced when acetate was present and repressing carbon sources were absent. Although glucose represses expression of the aceBA operon, repression does not seem to be mediated solely by cyclic AMP-cyclic AMP receptor protein complex. Mutants with altered regulation of the aceBA operon were isolated.

International audience; The flow of isocitrate through the glyoxylate bypass in Escherichia coli is regulated via the phosphorylation-dephosphorylation of isocitrate dehydrogenase mediated by a bifunctional enzyme: isocitrate dehydrogenase kinase/phosphatase. The aceK gene coding for this enzyme is part of the polycistronic ace operon, which also includes the aceB and aceA genes coding, respectively, for malate synthase and isocitrate lyase, the two glyoxylate bypass enzymes. The complete nucleotide sequence of a 2,214-base-pair DNA fragment containing the aceK gene and its 5' flanking region has been determined. In vivo experiments based on gene expression in a minicell system and protein fusion with beta-galactosidase, as well as in vitro assays with a plasmid-directed transcription-translation coupled system, have shown that the aceK gene extends over 1,731 nucleotides encoding a 66,528-dalton protein. The 5' flanking region presents an unusual intercistronic structural pattern consisting of two consecutive long dyad symmetries, almost identical in sequence, which can yield very stable stem-loop units. These structures are probably responsible for the drastic downshifting in expression observed in acetate-grown bacteria between the aceK gene and the aceA gene located immediately upstream in the ace operon.The flow of isocitrate through the glyoxylate bypass in Escherichia coli is regulated via the phosphorylation-dephosphorylation of isocitrate dehydrogenase mediated by a bifunctional enzyme: isocitrate dehydrogenase kinase/phosphatase. The aceK gene coding for this enzyme is part of the polycistronic ace operon, which also includes the aceB and aceA genes coding, respectively, for malate synthase and isocitrate lyase, the two glyoxylate bypass enzymes. The complete nucleotide sequence of a 2,214-base-pair DNA fragment containing the aceK gene and its 5' flanking region has been determined. In vivo experiments based on gene expression in a minicell system and protein fusion with beta-galactosidase, as well as in vitro assays with a plasmid-directed transcription-translation coupled system, have shown that the aceK gene extends over 1,731 nucleotides encoding a 66,528-dalton protein. The 5' flanking region presents an unusual intercistronic structural pattern consisting of two consecutive long dyad symmetries, almost identical in sequence, which can yield very stable stem-loop units. These structures are probably responsible for the drastic downshifting in expression observed in acetate-grown bacteria between the aceK gene and the aceA gene located immediately upstream in the ace operon.

In Escherichia coli, a single operon encodes the metabolic and regulatory enzymes of the glyoxylate bypass. The metabolic enzymes, isocitrate lyase and malate synthase, are expressed from aceA and aceB, and the regulatory enzyme, isocitrate dehydrogenase kinase/phosphatase, is expressed from aceK. We cloned this operon and determined its functional map by deletion analysis. The order of the genes in this operon is aceB-aceA-aceK, with aceB proximal to the promoter, consistent with the results of previous experiments using genetic techniques. The promoter was identified by S1 nuclease mapping, and its nucleotide sequence was determined. Isocitrate lyase and malate synthase were readily identified by autoradiography after the products of the operon clone were labeled by the maxicell procedure and then resolved by electrophoresis. In contrast, isocitrate dehydrogenase kinase/phosphatase, expressed from the same plasmid, was undetectable. This observation is consistent with a striking downshift in expression between aceA and aceK.

Acyl carrier protein (ACP coli) was isolated from commercially grown Escherichia coli B and was acetylated by chemical methods. Biological activity of the synthesized acetyl-ACP coli was checked in an in vitro fatty acid-synthesizing system isolated from E. coli B. Since acetyl-ACP is preferred over acetyl-coenzyme A (CoA) as a substrate in these reactions, the possibility that it may substitute for acetyl-CoA in biosynthetically and oxidatively important cellular pathways (glyoxylate and Krebs cycles, respectively) was examined. Acetyl-ACP was tested for substrate activity with the enzyme of each cycle which has been found to utilize acetyl-CoA. Crystalline citrate synthase (EC 4.1.3.7) of porcine origin (Calbiochem) was found to be inactive with acetyl-ACP coli, which acted neither as a substrate nor as an inhibitor in the presence of acetyl-CoA. Malate synthase (EC 4.1.3.2) of the acetate type was isolated from acetate-grown cells of a mutant of E. coli K-12 (VGD 3 H 5 ) and was also found to be inactive with acetyl-ACP coli. The significance of these results and of the recent discovery of another phospho-pantetheine-containing protein are discussed.

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora , isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.

The control of malate synthase formation in a fumaric acid-producing strain of Rhizopus nigricans has been found to be similar in most respects to that of isocitrate lyase, the companion enzyme of the glyoxylate bypass. A basal level is formed in a casein hydrolysate medium, which is repressed by glucose. Utilization of glucose during growth results in relief of glucose repression. Any factor which stimulates growth promotes relief of glucose repression by enhancing the incorporation of repressor metabolites derived from glucose into cell material. Thus, malate synthase formation was enhanced in glucose-containing media by the addition of zinc, or by an increase of the concentration of available nitrogen source in a synthetic medium. Both acetate and glycolate acted as apparent inducers of malate synthase, with glycolate the more effective of the two when added alone. Acetate induction was enhanced by Zn ++ , however, whereas induction by glycolate was unaffected. This supports the concept that acetate stimulates formation of glyoxylate bypass enzymes by a derepression mechanism, whereas glycolate or a product derived from it acts directly as an inducer. Moreover, it is indicated that the malate synthases induced by acetate and glycolate are separate and distinct, as has been shown in Escherichia coli .

Cells of Azotobacter vinelandii are specifically induced to encyst by β-hydroxybutyrate (BHB). The process of differentiation, which occurs over a period of 36 h, was characterized by an ordered sequence of biochemical events. Upon initiation of encystment, nitrogen fixation and glucose-6-phosphate dehydrogenase activities decreased immediately to very low levels. This was followed by an increase in the specific activities of BHB dehydrogenase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase first at 3 h and then again at 21 h. The peak activity of fructose 1,6-diphosphate aldolase occurred at 6 h, and the enzyme activity then decreased gradually. Fructose 1,6-diphosphatase had peak activities at 9 and 27 h. Deoxyribonucleic acid synthesis ceased just prior to the final cell division at 4 to 6 h, but ribonucleic acid synthesis continued until the 12th h. From labeling studies and the appearance of new enzyme activities, it appeared that protein synthesis continued throughout encystment.

Mutant strains of Escherichia coli K-12 were shown to be impaired in their ability to assimilate glycolate- 2 - 14 C . One strain (Glc-103) has lost the ability to oxidize glycolate; another strain (Glc-102) was relatively impermeable to the compound. A third strain (Glc-104) had undergone a similar loss in permeability, and, in addition, was deranged in the synthesis of either glyoxylate reductase or malate synthase G.