Regulation of Glyoxylate Metabolism in Escherichia coli K-12

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.     

Regulation of Thymidine Metabolism in Escherichia coli K-12: Studies on the Inducer and the Coordinateness of Induction of the Enzymes

A study was made of the regulation of three enzymes that act sequentially in the metabolism of thymidine in Escherichia coli K-12. Under a variety of conditions, two of the enzymes, thymidine phosphorylase and deoxyribose-5-phosphate aldolase, were found to be synthesized coordinately. However, the third enzyme, phosphodeoxyribomutase, was synthesized noncoordinately with the other two enzymes under the same conditions. In addition, the mutase could be fully induced, whereas basal levels of the phosphorylase and the aldolase were maintained. These findings indicate that two operons comprise the genes concerned with the reversible pathway leading from thymidine to acetaldehyde and glyceraldehyde-3-phosphate. In addition to thymidine, it was found that acetaldehyde was an external inducer of these enzymes. The results of induction experiments performed on wild-type cells and mutants defective in the mutase or the aldolase, with thymidine or acetaldehyde as exogenous inducers, strongly suggest that deoxyribose-5-phosphate is more proximal to the intracellular inducer than is thymidine, deoxyribose-1-phosphate, or acetaldehyde.     

Two proline porters in Escherichia coli K-12

Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus.     

Regulation of the Escherichia coli K-12 uvrB operon

The UV light inducibility of the uvrB operon of Escherichia coli K-12 was previously demonstrated by exploiting a strain in which the gene for the enzyme beta-galactosidase was inserted into the uvrB operon. This insert is now shown to be located within the structural gene for the uvrB enzyme, leaving the regulatory sequences of the operon intact. Analyses to quantitate the induction of this system show that derepression of the operon is first detectable 5 min after UV exposure, with the rate of synthesis increasing to four to six times the uninduced rate during the subsequent 30 min. Induction is unaffected by mutations in other components of nucleotide excision repair. The control of uvrB was found to result from direct repression by the lexA gene product, with the recA gene product playing an indirect role. Nucleotide excision repair thus seems to be part of the SOS response.     

Regulation of enterochelin synthetase in Escherichia coli K-12

Enterochelin synthetase activity is controlled by both repression and feed-back inhibition mechanisms. Inclusion of iron in growth media results in synthesis of all four (D, E, F and G) components of enterochelin synthetase being repressed. The specific inhibition of L-serine activation (partial reaction catalyzed by the F component) by the end products, ferric-enterochelin and 2,3-dihydroxybenzoylserine, is shown to inhibit overall enterochelin synthetase activity.     

Isolation of Transducing Bacteriophages for the Histidine and Isoleucine-Valine Operons in Escherichia coli K-12

In vitro studies have been of great value in elucidating the mechanism of the regulation of several bacterial operons. To obtain a deoxyribonucleic acid preparation enriched for the histidine (his) and for the isoleucine-valine (ilv) operons, we have isolated bacteriophages carrying the his and the ilv regions of the Escherichia coli chromosome. Transposition of the his operon to a site close to the att80 region of the E. coli chromosome has been carried out selecting for integration of a temperature-sensitive F'his(+) in the tonB locus. This transposed strain has been lysogenized with phi80i(lambda). Upon induction of the lysogen, His(+) transductants have been isolated, which, on further induction give rise to HFT (high frequency of transduction) lysates. Preliminary characterization of the transducing phage is reported. The ilv operon, carried on an F' particle, has been fused to an episome carrying the att80 region. The fused episome has been lysogenized with phi80i lambdat68. Upon induction of the lysogen, Ilv(+) transductants have been isolated which on further induction give rise to HFT lysates.     

Mutations Affecting Amino Sugar Metabolism in Escherichia coli K-12

The genetic loci determining N-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase have been located at min 16 on the Escherichia coli K-12 genetic map.     

Genetic Control of the 2-Keto-3-Deoxy-d-Gluconate Metabolism in Escherichia coli K-12: kdg Regulon

2-Keto-3-deoxy-gluconate (KDG), an intermediate of the hexuronate pathway in Escherichia coli K-12, is utilized as the sole carbon source only in strains derepressed for the specific KDG-uptake system. KDG is metabolized to pyruvate and glyceraldehyde-3-phosphate via the inducible enzymes KDG-kinase and 2-keto-3-deoxy-6-phosphate-gluconate (KDPG) aldolase. However, another inducible pathway, where the KDG is the branch point, has been demonstrated. Genetic studies of the KDG degradative pathway reported in this paper led to the location of KDG kinase-negative and pleiotropic constitutive mutations. The kdgK locus, presumably the structural gene of the kinase, occurs at min 69 and is co-transducible with xyl. The mutants, simultaneously constitutive for the uptake, kinase, and aldolase, define a kdgR locus at min 36 between the co-transducible markers kdgA and oldD. As to the nature of the control exerted by the kdgR product, we have shown the following. (i) Thermosensitive mutants of the kdgR locus are inducible at low temperature but derepressed at 42 C for the three operons—kdgT (transport system), kdgK, and kdgA (KDPG aldolase). (ii) The kdgR⁺ allele is dominant to the kdgR constitutive allele. (iii) A deletion in kdgA extending into the regulatory gene, kdgR, leads to a constitutive expression of the nondeleted operons—kdgT and kdgK. These properties demonstrate that the kdg regulon is negatively controlled by the kdgR product. It is presumed that differences in operator and in promotor structures could explain the strong decoordination, respectively, in the induction and catabolic repression, of these three enzymes activities.     

Regulation of lysine decarboxylase activity in Escherichia coli K-12

The biodegradative lysine decarboxylase of E. coli has been reported to attain a higher specific activity when grown to saturation in the presence of excess lysine under conditions of low pH and absence of aeration. In order to examine possible sources of the pH and anaerobic regulation, a series of isogenic strains of E. coli K-12 were constructed. The effects of cadR^-, fnr ^-, cya ^-, crp ^-and pgl ^-mutations on lysine decarboxylase expression were examined. Cultures were grown in a lysine supplemented rich medium at pH 5.5, pH 6.8, and pH 8.0 with and without aeration and the enzyme was assayed from log phase cultures. The results suggested that the pH and air responses were independent and that these known regulatory processes are not responsible for this regulation of the biodegradative lysine decarboxylase.     

Regulation of D-arabinose utilization in Escherichia coli K-12.

Studies involving lambda phage transduction of the D-arabinose utilization gene (dar+) in Escherichia coli K-12 indicated the product of this gene to be a transdominant activator. An apparent anomaly regarding this hypothesis exists in that a diploid recessive lysogen (lambda dar-/dar-) can spontaneously become capable of growth on D-arabinose.     

Metabolism of D-serine in Escherichia coli K-12: mechanism of growth inhibition

Without significant killing, d-serine at concentrations greater than 50 mug/ml inhibits growth in minimal media of mutants of Escherichia coli K-12 unable to form d-serine deaminase. The mutants eventually recover at lower concentrations. There is no evidence of d-serine toxicity in rich media. Toxicity is partially reversed by l-serine. d-Serine does not interfere with l-serine activation, one-carbon metabolism, or (Cronan, personal communication) formation of phosphatidylserine. Pizer (personal communication) finds, however, that it is a powerful feedback inhibitor of the first enzyme of l-serine biosynthesis. In the presence of l-serine, the residual toxicity is largely and noncompetitively over come by pantothenate, indicating that d-serine inhibits growth by affecting two targets: pantothenate biosynthesis and l-serine biosynthesis. l-Serine causes transient growth inhibition in E. coli K-12. Contaminating l-serine in d-serine preparations contributes to the d-serine inhibitory response.     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.     

Degradation of missense mutant beta-galactosidase proteins in Escherichia coli K-12.

Summary Ten out of 43 missense mutations in the lacZ gene of Escherichia coli gave rise to polypeptide chains that were degraded in vivo. While many of the mutants appeared to be fully or partially CRM^–, there appeared to be no obvious correlation between degradation, map position, altered subunit association and the half-life of the mutant proteins.     

Metabolism of arginine-specific messenger ribonucleic acid in Escherichia coli K-12.

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of the level of messenger RNA (mRNA) transcribed from seven of the nine genes of the arginine regulon of Escherichia coli K-12. The quantity of RNA complexing with each of the separated DNA strands of the argA, argF, argE, and argCBH operons carried on specialized transducing phages was measured. The derepressed:repressed ratio of mRNA formed in vivo was found to vary between about 3 and 4 when measured by hybridization to DNA isolated from specialized transducing phages carrying the argA, argE, argCBH, argF, and argI operons.