Glycerol kinase, the pacemaker for the dissimilation of glycerol in Escherichia coli

The activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. A mutant strain producing a glycerol kinase resistant to inhibition by fructose-1,6-diphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon and energy. The amount of intracellular fructose-1,6-diphosphate was determined for wild-type cells growing exponentially on glycerol. The water content of such cells was also determined, allowing calculation of the intracellular concentration of fructose-1,6-diphosphate. This value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type glycerol kinase. The desensitization of glycerol kinase to feedback inhibition also enhances the power of glycerol to exert catabolite repression, both on the enzymes of the glycerol system itself and on those of the lactose system. However, desensitization of glycerol kinase alone does not eliminate the phenomenon of diauxic growth in a glucose-glycerol medium. Biphasic growth in such a medium is abolished if the altered enzyme is produced constitutively. The constitutive production of the mutant kinase at high levels, however, renders the cells vulnerable to glycerol. Thus, when the cells have been grown on a carbon source with a low power for catabolite repression, e.g., succinate, sudden exposure to glycerol leads to overconsumption of the nutrient and cell death.     

Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli.

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.     

Cloning of Streptomyces griseus and Streptomyces lividans genes for glycerol dissimilation

Streptomyces lividans gyl DNA (for glycerol utilisation) was cloned by complementation of a Streptomyces coelicolor gyl mutant. Restriction mapping showed that the cloned DNA was highly homologous (perhaps 99%) to S. coelicolor gyl DNA. Using phage-mediated mutational cloning, an internal fragment of the S. coelicolor gyl operon was used to generate a gyl mutant of S. lividans, which subsequently served as recipient in the cloning of gyl DNA from S. griseus. A 7.5-kb SstI-generated fragment of S. griseus DNA was obtained which, as judged by analysis of restriction sites, was only perhaps 87% homologous with the S. coelicolor gyl operon. The cloned S. griseus DNA appears to contain intact gylA and gylB genes and probably also an upstream gene related to the putative gyl regulatory '0.9-kb' gene of S. coelicolor. Cloning of the fragment on a high-copy-number vector in S. lividans did not lead to high levels of the enzymes encoded by gylA and gylB. The S. griseus gylA and gylB genes were not detectably expressed in Escherichia coli glp mutants.     

Experimental determination of control by the H+-ATPase inEscherichia coli

Strains carrying deletions in theatp genes, encoding the H⁺-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of anatp deletion mutant was surprisingly high (some 75–80% of wild-type growth rate). The rate of glucose and oxygen consumption of these mutants was increased compared to the wild-type rates. In order to analyze the importance of the H⁺-ATPase at its physiological level, the cellular concentration of H⁺-ATPase was modulated around the wild-type level, using genetically manipulated strains. The control coefficient by the H⁺-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wild-type concentration of H⁺-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H⁺-ATPase on the catabolic fluxes, including respiration, was negative at the wild-type H⁺-ATPase level. Moreover, the turnover number of the individual H⁺-ATPase enzymes increased as the H⁺-ATPase concentration was lowered. The negative control by the H⁺-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some extent, explain the zero control by the H⁺-ATPase onE. coli growth rate.     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

Characterization of a novel cellobiase fromBacillus subtilis and expression of its structural gene inEscherichia coli

Summary A strain ofBacillus subtilis was found to produce a cellobiase resistant to catabolic repression by glucose. When the structural gene encoding cellobiase was cloned and expressed inEscherichia coli, the enzyme produced was resistant to repression by glucose.     

Localization of L-asparaginase inEscherichia coli

The localization ofl-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) EC-2 isoenzyme was studied inEscherichia coli ATCC 9637 grown under conditions of moderate aeration. The enzyme was determined in cell fractions obtained by fraction centrifugation of lysed spheroplasts. When the synthesis of the enzyme was induced byl-asparagine, its amount in the cytoplasmic fraction at the beginning of the induction exceeded as much as five times that in uninduced cells, attaining up to 20% of the total activity. In the course of growth of the culture this activity decreased gradually to zero. The membrane fraction of induced cells contained considerable amount of EC-2l-asparaginase which, at the beginning of the induction, reached up to 6% ot the total enzymic activity; in membrane fraction of control cells the activity was close to zero. The results indicate a relationship of cell structures to thel-asparagine-induced synthesis of the enzyme.     

Expression of a human proenkephalin a cDNA inEscherichia coli

A 1000 base pair cDNA coding for the entire human proenkephalin A(proA) polypeptide was subcloned into the multifunctional pMPV 2911/ME. coli vector. The recombinant plasmid was found to express an approximately 30 kDa prohormone, which was recognized by a Met-Arg⁶-Phe² antibody, directed against the C-terminal part of the enkephalin A prohormone. The expression of human proenkephalin A cDNA should thus permit the rapid purification of unfused recombinant enkephalin A prohormone, which itself may provide a model substrat to identify endoproteolytic processing activities.     

Evidence for the existence of a novel component of biological water stress (anhydrotic stress) inEscherichia coli

The pathways for biosynthesis of pyrimidines, L-arginine and the polyamines are intimately interrelated in many microorganisms. We discovered in this study that growth of wild-typeEscherichia coli in low-water-activity minimal media is inhibited by the addition of uracil. Uracil sensitivity was observed irrespective of whether the dissolved solute(s) contributing to decreased water activity was ionic (e.g. NaCl, K2SO4), nonionic and impermeable (e.g. sucrose), nonionic and freely permeable (e.g. glycerol), or any mixture of these types. A mutant resistant to such growth inhibition was isolated and was shown to harbour a bradytrophic mutation inargA, the gene encoding the first step in the L-arginine biosynthetic pathway. Mutations inargR, whose product is the aporepressor of the same pathway, or exogenous supplementation with L-arginine or L-citrulline, also conferred resistance to uracil inhibition in low-water-activity media. A similar uracil-sensitivity phenotype, which was reversible byargA, argR, or L-arginine addition, was exhibited even in media with a more moderate reduction in water activity in two different situations: for aspeC mutant (which is defective in the enzyme ornithine decarboxylase required for biosynthesis of the polyamines) and for the wild-type strain in media additionally supplemented with L-ornithine. On the basis of these observations, we propose a model in which high cytoplasmic levels of the intermediary metabolite L-ornithine are inhibitory to growth ofE. coli in media of low water activity. Our results also provide the first evidence for the existence of a third component of physiological water stress, which is elicited by both impermeable and permeable dissolved solutes (the other two known components are ionic stress, which is elicited only by ionic solutes, and osmotic stress, which is elicited only by impermeable solutes either ionic or nonionic). We propose the term anhydrotic stress to refer to this novel component of water stress.     

Uptake and dissimilation of glycerol by wild type and glycerol nonutilizing strains of Neurospora crassa

Summary Seven mutant strains defective for utilization of glycerol, glyceraldehyde or dihydroxyacetone were isolated. One strain was deficient for NAD-linked glycerol-3-phosphate dehydrogenase, two for glycerol kinase, and four had no detected enzymatic deficiency, although one of the latter strains was deficient in glycerol uptake. Glycerol uptake was increased by incubation in glycerol, glycerol-3-phosphate, erythritol, and propanediol, and was protein-mediated below 0.14 mM glycerol, but at higher concentrations free diffusion predominated. Glycerol uptake was decreased by cycloheximide and was more sensitive to sodium azide than to iodoacetate.     

Genetic control of flocculation inEscherichia coli

Summary Escherichia coli cells form flocs or aggregates by overproducing type 1 pili. When thepil operon is placed under the control of atac orlac promoter-operator sequence, the bacterial cells can be induced to form flocs by adding isopropyl--d-thiogalactopyranoside to the culture medium. This phenomenon of genetically induced flocculation can aid in the downstream processing of biological products. This paper describes the construction of two artificially controlled plasmids which cause cell flocculation. Cell aggregates 50 m in mean diameter were obtained 1 h after the cells were induced.     

Partial purification of alpha-hemolysin inEscherichia coli

The present paper describes isolation and purification ofa-hemolysin ofEscherichia coli. The optimum production medium was found to be the Todd—Hewitt broth. Out of thirteen fractions obtained after separation on Sephadex G-200, two fractions possessed the highest relative specific activity.     

Expression of theunc genes inEscherichia coli

Theunc (or atp) operon ofEscherichia coli comprises eight genes encoding the known subunits of the proton-translocating ATP synthase (H⁺-ATPase) plus a ninth gene (uncI) of unknown function. The subunit stoichiometry of the H⁺-ATPase ( ₃₃₁₁₁a₁b₂c_10–15) requires that the respectiveunc genes be expressed at different rates. This review discusses the experimental methods applied to determining how differential synthesis is achieved, and evaluates the results obtained. It has been found that the primary level of control is translational initiation. The translational efficiencies of theunc genes are determined by primary and secondary mRNA structures within their respective translational initiation regions. The respective rates of translation are matched to the subunit requirements of H⁺-ATPase assembly. Finally, points of uncertainty remain and experimental strategies which will be important in future work are discussed.     

Experimental evolution of a novel sexually antagonistic allele

Evolutionary conflict permeates biological systems. In sexually reproducing organisms, sex-specific optima mean that the same allele can have sexually antagonistic expression, i.e. beneficial in one sex and detrimental in the other, a phenomenon known as intralocus sexual conflict. Intralocus sexual conflict is emerging as a potentially fundamental factor for the genetic architecture of fitness, with important consequences for evolutionary processes. However, no study to date has directly experimentally tested the evolutionary fate of a sexually antagonistic allele. Using genetic constructs to manipulate female fecundity and male mating success, we engineered a novel sexually antagonistic allele (SAA) in Drosophila melanogaster. The SAA is nearly twice as costly to females as it is beneficial to males, but the harmful effects to females are recessive and X-linked, and thus are rarely expressed when SAA occurs at low frequency. We experimentally show how the evolutionary dynamics of the novel SAA are qualitatively consistent with the predictions of population genetic models: SAA frequency decreases when common, but increases when rare, converging toward an equilibrium frequency of ～8%. Furthermore, we show that persistence of the SAA requires the mating advantage it provides to males: the SAA frequency declines towards extinction when the male advantage is experimentally abolished. Our results empirically demonstrate the dynamics underlying the evolutionary fate of a sexually antagonistic allele, validating a central assumption of intralocus sexual conflict theory: that variation in fitness-related traits within populations can be maintained via sex-linked sexually antagonistic loci.     

A novel pathway for lipid biosynthesis: the direct acylation of glycerol.

The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions.