Interrelationships between the enzymes of ethanolamine metabolism in Escherichia coli

The activities of the enzymes ethanolamine ammonia-lyase, CoA-dependent and CoA-independent aldehyde dehydrogenases, and isocitrate lyase were assayed in Escherichia coli which had been grown on various sources of carbon and nitrogen. Induction of ethanolamine ammonia-lyase and of maximal levels of both aldehyde dehydrogenases required the concerted effects of ethanolamine and vitamin (or coenzyme) B12. Molecular exclusion chromatography revealed that, in the absence of one or both co-inducers, two repressible isoenzymes of CoA-dependent aldehyde dehydrogenase (mol. wts 900000 and 120000) were produced, these being replaced by two inducible isoenzymes (mol. wts 520000 and 370000) in the presence of both co-inducers. A similar inducible repressible series of isoenzymes was also observed for CoA-independent aldehyde dehydrogenase. No evidence was found for structural relationships between ethanolamine ammonia-lyase, CoA-dependent aldehyde dehydrogenase and CoA-independent aldehyde dehydrogenase, but mutant and physiological studies demonstrated that the induction of the first two enzymes is under common control. Evidence is presented for the operation of a previously unreported pathway of ethanolamine metabolism in E. coli.     

A pathway for putrescine catabolism in Escherichia coli

Escherichia coli mutants able to grow in putrescine have been isolated from gamma-aminobutyrate mutants. These mutants show putrescine-alpha-ketoglutarate transaminase and gamma-aminobutyraldehyde dehydrogenase activities. Both enzymes have been characterized, the first of them showing an apparent Km for putrescine of 22.5 microM and the second an apparent Km of 37 microM for NAD and 18 microM for delta-1-pyrroline; the optimum pH values were 7.2 and 5.4, respectively, for the two enzymes.     

A regulatory mutant affecting the synthesis of enzymes involved in the catabolism of nucleosides in Escherichia coli

Summary A regulatory mutant which leads to constitutive synthesis of enzymes involved in catabolism of nucleosides is described. It is unlinked to the structural genes whose activity is affected. The gene concerned is designated nucR. The amount of thymine required for growth (colony formation) of thy ^– strains is affected by the nucR mutation. The amount required by a thy ^– drm ^–strain is reduced about four fold if it carries the constitutivity mutation. The amount required by a thy ^– drm ⁺strain is increased at least two fold. These differences in nutritional requirement provide a method for selecting constitutives from non-constitutives and vice versa.Abbreviations Rib-1-P Ribose-1-phosphate - dRib-1-P deoxyribose-1-phosphate - Rib-5-P Ribose-5-phosphate - dRib-5-P deoxyribose-5-phosphate - Pi inorganic phosphate     

A map of four genes specifying enzymes involved in catabolism of nucleosides and deoxynucleosides in Escherichia coli

Summary Four genes specifying the enzymes thymidine phosphorylase, purine nucleoside phosphorylase, deoxyribomutase and deoxyriboaldolase were mapped by transduction with phage P1. All pairs show greater than 90 per cent co-transduction. The gene order was found to be dra-tpp-drm-pup, and the gene cluster was shown to lie between the hsp and ser B loci on the chromosome map of Escherichia coli.This work is supported by Grant No. B/SR/3113 from the Science Research Council.     

An operator constitutive mutant affecting the synthesis of two enzymes involved in the catabolism of nucleosides in Escherichia coli

Summary A new type of mutant of mutant of Escherichia coli that synthesises thymidine phosphorylase constitutively has been isolated and characterised. The mutation leading to constitutivity is located to the left of the gene specifying deoxyriboaldolase. The mutation is cis dominant in its effect on thymidine phosphorylase activity and therefore believed to be a mutation of the operator-constitutive type. The specific activity of purine nucleoside phosphorylase is not affected by the mutation indicating that the gene specifying this enzyme is located in a different operon from that containing the genes specifying thymidine phosphorylase and deoxyriboaldolase.     

Ribitol and D-arabitol catabolism in Escherichia coli.

In Escherichia coli C, the catabolism of the pentitols ribitol and D-arabitol proceeds through separate, inducible operons, each consisting of a dehydrogenase and a kinase. The ribitol operon is induced in response to ribulose, and the D-arabitol operon is induced in response to D-arabitol. Each operon is under negative control. The genes of the ribitol and D-arabitol operons are very closely linked and lie in a mirror image arrangement, rtlB-rtlA-rtlC-atlC-atlA-atlB, between metG and his on the E. coli chromosome.     

A model for the initiation of replication in Escherichia coli

The role of the protein DnaA as the principal control of replication initiation is investigated by a mathematical model. Data showing that DnaA is growth rate regulated suggest that its concentration alone is not the only factor determining the timing of initiation. A mathematical model with stochastic and deterministic components is constructed from known experimental evidence and subdivides the total pool of DnaA protein into four forms. The active form, DnaA.ATP, can be bound to the origin of replication, oriC, where it is assumed that a critical level of these bound molecules is needed to initiate replication. The active form can also exist in a reserve pool bound to the chromosome or a free pool in the cytoplasm. Finally, a large inactive pool of DnaA protein completes the state variables and provides an explanation for how the DnaA.ATP form could be the principal controlling element in the timing of initiation. The fact that DnaA protein is an autorepressor is used to derive its synthesis rate. The model studies a single exponentially growing cell through a series of cell divisions. Computer simulations are performed, and the results compare favorably to data for different cell cycle times. The model shows synchrony of initiation events in agreement with experimental results.     

Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes

This study investigates the effect of overexpression of key glycolytic enzymes exhibiting either native or alternative allosteric regulation on glucose bioconversion by resting Escherichia coli cells previously engineered for ethanol production. Homologous and heterologous pyruvate kinases (Pyk) and phosphofructokinases (Pfk) were individually and simultaneously overexpressed. Overexpression of the E. coli Pfk led to a shift from ethanol to lactate formation (three-fold above the control level) while overexpression of Pyks accelerated lactate formation two-fold with less reduction in ethanol formation. Further increase in lactate formation (five-fold above the control level) resulted from overexpression of Pfk from Lactobacillus bulgaricus which, unlike the E. coli Pfk, is not allosterically regulated by either phosphoenolpyruvate or ADP. These effects on the carbon flux distribution were accompanied by significant changes in the intracellular concentrations of several glycolytic intermediates. Increased Pfk levels led primarily to reduced levels of hexose phosphates. Increased Pyk activity resulted in more complex changes which were different for overexpressed native Pyk and for overexpressed Bacillus stearothermophilus Pyk, which differs from E. coli Pyk in lacking activation by fructose 1,6-diphosphate, but is allosterically activated by AMP and ribose 5-phosphate. Simultaneous overexpression of native Pfk and Pyk caused a Pfk-overexpression-like phenotype with lower levels of hexose phosphates and further increased lactate formation (nine-fold above the control level). The flux data demonstrate that overexpression of even single enzymes early in a central pathway can increase the fluxes to a particular metabolic product, although it may not affect the glucose uptake rate.     

A generic system for the Escherichia coli cell-surface display of lipolytic enzymes

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.     

2-Phenylethylamine catabolism by Escherichia coli K12

Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.     

Pathway and enzyme redundancy in putrescine catabolism in Escherichia coli

Putrescine as the sole carbon source requires a novel catabolic pathway with glutamylated intermediates. Nitrogen limitation does not induce genes of this glutamylated putrescine (GP) pathway but instead induces genes for a putrescine catabolic pathway that starts with a transaminase-dependent deamination. We determined pathway utilization with putrescine as the sole nitrogen source by examining mutants with defects in both pathways. Blocks in both the GP and transaminase pathways were required to prevent growth with putrescine as the sole nitrogen source. Genetic and biochemical analyses showed redundant enzymes for γ-aminobutyraldehyde dehydrogenase (PatD/YdcW and PuuC), γ-aminobutyrate transaminase (GabT and PuuE), and succinic semialdehyde dehydrogenase (GabD and PuuC). PuuC is a nonspecific aldehyde dehydrogenase that oxidizes all the aldehydes in putrescine catabolism. A puuP mutant failed to use putrescine as the nitrogen source, which implies one major transporter for putrescine as the sole nitrogen source. Analysis of regulation of the GP pathway shows induction by putrescine and not by a product of putrescine catabolism and shows that putrescine accumulates in puuA, puuB, and puuC mutants but not in any other mutant. We conclude that two independent sets of enzymes can completely degrade putrescine to succinate and that their relative importance depends on the environment.     

Regulation of the early steps of 3-phenylpropionate catabolism in Escherichia coli

Microbial catabolism of phenylpropanoid compounds plays a key role in the degradation of aromatic molecules originating from the degradation of proteins and plant constituents. In this study, the regulation of the early steps in the utilisation of 3-phenylpropionate, a phenylpropanoid compound, was investigated. Expression of the hcaA gene product, which is involved in 3-phenylpropionate catabolism in Escherichia coli, was positively regulated by HcaR, a regulatory protein similar to members of the LysR regulators family. Remarkably, the expression of hcaA in the presence of 3-phenylpropionate was sharply and transiently induced at the end of the exponential growth phase. This occurred in a rpoS-independent manner. This transient induction was also mediated by HcaR. The expression of this positive regulator is negatively autoregulated, as for other members of the LysR family. The expression of hcaR is strongly repressed in the presence of glucose. Glucose-dependent repression of hcaR expression could only be partially overcome by adding exogenous cAMP.     

A common pathway for the activation of several molybdoenzymes in Escherichia coli K12

Three molybdoenzymes, nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase which form part of different systems, have been studied in a parental strain of Escherichia coli K12. When the organism is grown in the presence of 10 mM tungstate, these three enzymes are present in an inactive form which may be activated in vivo by the addition of 1 mM sodium molybdate. The mixing of soluble fractions from chlA and chlB mutants grown under the appropriate conditions leads to the activation of nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase. The activation of each enzyme is maximal when the mutants are grown under conditions that lead to the induction of that enzyme in the wild-type strain. The employment of purified proteins, the association factor FA and the Protein PA, which are presumed to be the products of the chlA and chlB genes, has shown that these proteins are responsible for the activation of the three enzymes during the complementation process.