David C. LaPorte

Global regulatory mutations in csrA and rpoS cause severe central carbon stress in Escherichia coli in the presence of acetate

The csrA gene encodes a small RNA-binding protein, which acts as a global regulator in Escherichia coli and other bacteria (T. Romeo, Mol. Microbiol. 29:1321-1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of csrA in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of >/=25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that csrA positively regulates acs (acetyl-coenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.

Sensitivity of Metabolic Fluxes to Covalent Control

Publisher Summary This chapter focuses on the sensitivity of metabolic fluxes to covalent control. A major goal in current biological research is to achieve a thorough understanding of the control of metabolic processes. Recent developments suggest that the pathology resulting from chromosomal aneuploidy, oncogene products, and protein overproduction may ultimately be understood in terms of the sensitivity of metabolic processes to control. To obtain a better understanding of the factors controlling the flow through the branch point, many techniques have been developed to measure the rates of carbon flux through the Krebs cycle, the glyoxylate shunt, and ancillary reactions. A key step in the understanding of regulation can be achieved by comparing in vivo fluxes with values calculated from the in vitro constants of the respective enzymes. The partitioning of the flux through the branch point of the Krebs cycle and the glyoxylate shunt is seen to be highly sensitive to control. The control has the interesting feature that the enzyme most affected, the lyase, is not subject to direct control. Instead, on addition of glucose, isocitrate dehydrogenase is directly regulated by dephosphorylation and the rate of production of isocitrate is decreased. As a consequence, the flux through the alternate pathway is drastically reduced, dropping almost to zero.

Response of a yeast glycogen synthase gene to stress.

In the yeast Saccharomyces cerevisiae, glycogen synthase is encoded by two genes: GSY1 and GSY2. The activity of the enzymes increases as cultures enter the stationary phase of growth. Using a GSy2::lacZ fusion gene, we have demonstrated that the increase in glycogen synthase activity resulted, at least in part, from an increase in the level of the protein rather than simply from a change in its phosphorylation state. Northern blot analysis showed a parallel increase in the level of the GSY2 mRNA, which is consistent with transcriptional activation of GSY2. Deletion analysis identified three regions upstream of GSY2 which are involved in GSy2 expression: regions A (‐390 to ‐347 relative to the start of translation), B (‐252 to ‐209) and C (‐209 to ‐167). Region A or C independently activated expression of GSY2. In contrast, region B alone yielded only modest expression. Expression of GSY2 is induced by growth to stationary phase, heat shock or nitrogen starvation. Response to these stressors is mediated by elements within regions A and C. These elements appear to be related to the stress‐response elements found in other stress‐responsive genes.

Relationship between changes in the calcium dependent regulatory protein and adenylate cyclase during viral transformation

Abstract The levels of the calcium dependent regulatory protein in transformed chicken embryo fibroblasts are higher both in soluble fractions and membrane fractions compared to untransformed cells. The kinetics for changes in the calcium dependent regulatory protein, hexose transport, and adenylate cyclase were compared using a temperature sensitive mutant of Rous sarcoma virus. Decreases in adenylate cyclase activity and increased hexose transport accompanying transformation occurred with half-lives of approximately 7 to 8 hours. Increases in the calcium dependent regulatory protein occurred much slower with a half-life of seventeen hours. It is concluded that the increase in calcium dependent regulatory protein levels is a late event during viral transformation and that the decline in adenylate cyclase activity cannot be due to changes in the amount of calcium dependent regulatory protein.

GLC3 and GHA1 of Saccharomyces cerevisiae are allelic and encode the glycogen branching enzyme

In the yeast Saccharomyces cerevisiae, glycogen serves as a major storage carbohydrate. In a previous study, mutants with altered glycogen metabolism were isolated on the basis of the altered iodine-staining properties of colonies. We found that when glycogen produced by strains carrying the glc-1p (previously called gha1-1) mutation is stained with iodine, the absorption spectrum resembles that of starch rather than that of glycogen, suggesting that this mutation might reduce the level of branching in the glycogen particles. Indeed, glycogen branching activity was undetectable in extracts from a glc3-1p strain but was elevated in strains which expressed GLC3 from a high-copy-number plasmid. These observations suggest that GLC3 encodes the glycogen branching enzyme. In contrast to glc3-1p, the glc3-4 mutation greatly reduces the ability of yeast to accumulate glycogen. These mutations appear to be allelic despite the striking difference in the phenotypes which they produce. The GLC3 clone complemented both glc3-1p and glc3-4. Deletions and transposon insertions in this clone had parallel effects on its ability to complement glc3-1p and glc3-4. Finally, a fragment of the cloned gene was able to direct the repair of both glc3-1p and glc3-4. Disruption of GLC3 yielded the glycogen-deficient phenotype, indicating that glycogen deficiency is the null phenotype. The glc3-1p allele appears to encode a partially functional product, since it is dominant over glc3-4 but recessive to GLC3. These observations suggest that the ability to introduce branches into glycogen greatly increases the ability of the cell to accumulate that polysaccharide. Northern (RNA) blot analysis identified a single mRNA of 2,300 nucleotides that increased in abundance ca. 20-fold as the culture approached stationary phase. It thus appears that the expression of GLC3 is regulated, probably at the level of transcription.

Expression of the yeast glycogen phosphorylase gene is regulated by stress-response elements and by the HOG MAP kinase pathway

Yeast glycogen metabolism responds to environmental stressors such as nutrient limitation and heat shock. This response is mediated, in part, by the regulation of the glycogen metabolic genes. Environmental stressors induce a number of glycogen metabolic genes, including GPH1, which encodes glycogen phosphorylase. Primer extension analysis detected two start sites for GPH1, one of which predominated. Sequences upstream of these sites included a possible TATA element. Mutation of this sequence reduced GPH1 expression by a factor of 10 but did not affect start site selection. This mutation also did not affect the relative induction of GPH1 upon entry into stationary phase. Three candidates for stress response elements (STREs) were found upstream of the TATA sequence. Mutation of the STREs showed that they were required for regulation of GPH1 expression in early stationary phase, and in response to osmotic shock and heat shock. These elements appeared to act synergistically, since the intact promoter exhibited 30‐fold more expression in stationary phase than the sum of that observed for each element acting independently. HOG1, which encodes a MAP kinase, has been implicated in control mediated by STREs. For GPH1, induction by osmotic shock depended on a functional HOG1 allele. In contrast, induction upon entry into stationary phase was only partially dependent on HOG1. Furthermore, the heat shock response, which can also be mediated by STREs, was independent of HOG1. These observations suggest that the GPH1 STREs respond to more than one pathway, only one of which requires HOG1. Copyright

Isocitrate Dehydrogenase Kinase/Phosphatase KINETIC CHARACTERISTICS OF THE WILD-TYPE AND TWO MUTANT PROTEINS

Isocitrate dehydrogenase (IDH) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In addition to the kinase and phosphatase activities, this protein catalyzes an intrinsic ATPase reaction. The initial velocity kinetics of these activities exhibited extensive similarities. IDH kinase and phosphatase both yielded intersecting double-reciprocal plots. In addition, we observed similar values for the kinetic constants describing interactions of the kinase and phosphatase with their protein substrates and the interactions of all three activities with ATP. In contrast, while the maximum velocities of IDH kinase and IDH phosphatase were nearly equal, they were 10-fold less than the maximum velocity of the ATPase. Although the IDH phosphatase reaction required either ATP or ADP, it was not supported by the nonhydrolyzable ATP analogue 5′-adenylyl imidodiphosphate. The kinetic properties of wild-type IDH kinase/phosphatase were compared with those of two mutant derivatives of this protein. The mutations in these proteins selectively inhibit IDH phosphatase activity. Inhibition of IDH phosphatase resulted from three factors: decreases in the maximum velocities, reduced affinities for phospho-IDH, and a loss of coupling between ATP and phospho-IDH. These mutations also affected the properties of IDH kinase, increasing the maximum velocities and decreasing the affinities for ATP and phospho-IDH. The intrinsic ATPase activities also exhibited reduced affinity for ATP. These results are discussed in the context of a model which proposes that all three activities occur at the same active site.

Locations of the Regulatory Sites for Isocitrate Dehydrogenase Kinase/Phosphatase

Isocitrate dehydrogenase (IDH)1 ofEscherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In this paper, we demonstrate that the effectors controlling these activities belong to two distinct classes that differ in mechanism and in the locations of their binding sites. NADPH and isocitrate are representative members of one of these effector classes. NADPH inhibits both IDH kinase and IDH phosphatase, whereas isocitrate inhibits only IDH kinase. Isocitrate can “activate” IDH phosphatase by reversing product inhibition by dephospho-IDH. Mutations in icd, which encodes IDH, had parallel effects on the binding of these ligands to the IDH active site and on their effects on IDH kinase and phosphatase, indicating that these ligands regulate IDH kinase/phosphatase through the IDH active site. Kinetic analyses suggested that isocitrate and NADPH prevent formation of the complex between IDH kinase/phosphatase and its protein substrate. AMP, 3-phosphoglycerate, and pyruvate represent a class of regulatory ligands that is distinct from that which includes isocitrate and NADPH. These ligands bind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of this enzyme. These effector classes can also be distinguished by the observation that mutant derivatives of IDH kinase/phosphatase expressed from aceK3 andaceK4 exhibited dramatic changes in their responses to AMP, 3-phosphoglycerate, and pyruvate but not to NADPH and isocitrate.

Introduction of single-copy sequences into the chromosome of Escherichia coli: application to gene and operon fusions

We have developed a general method for the introduction of any cloned sequence into the chromosome of Escherichia coli. This method employs an Hfr strain which carries a fragment of bla (the pBR322 gene imparting ampicillin resistance) between lacI and lacZ. Plasmid-borne inserts which are flanked by sequences from bla and lacZ can be introduced at this locus by homologous recombination. The isolation of recombinants is enhanced by selection for transfer of an integrated copy of the plasmid during conjugation. Once introduced into the chromosome, the inserted sequences can be transferred to other strains by conventional methods such as P1 transduction or conjugation. This method is suitable for the transfer of any cloned sequence to the chromosome and is particularly well suited to the construction of chromosomal gene and operon fusions with lacZ.