Evolution of a regulated operon in the laboratory

The evolution of new metabolic functions is being studied in the laboratory using the EBG system of E. coli as a model system. It is demonstrated that the evolution of lactose utilization by lacZ deletion strains requires a series of structural and regulatory gene mutations. Two structural gene mutations act to increase the activity of ebg enzyme toward lactose, and to permit ebg enzyme to convert lactose into allolactose, an inducer of the lac operon. A regulatory mutation increases the sensitivity of the ebg repressor to lactose, and permits sufficient ebg enzyme activity for growth. The resulting fully evolved ebg operon regulates its own expression, and also regulates the synthesis of the lactose permease.     

Regulation of Newly Evolved Enzymes. IV. Directed Evolution of the EBG Repressor

In Escherichia coli, the wild-type repressor of ebg (evolved beta-galactosidase) enzyme synthesis, specified by the ebgR+ gene, responds very weakly to lactulose (fructose-beta-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 X 10(-10) . EBGR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.     

Experimental Evolution of a New Enzymatic Function. II. Evolution of Multiple Functions for EBG Enzyme in E. COLI

The evolution of ebgo enzyme of Escherichia coli, an enzyme which is unable to hydrolyze lactose, lactulose, lactobionate, or galactose-arabinoside effectively, has been directed in successive steps so that the evolved enzyme is able to hydrolyze these galactosides effectively. I show that in order for a strain of E. coli with a lacZ deletion to evolve the ability to use lactobionate as a carbon source, a series of mutations must occur in the ebg genes, and that these mutations must be selected in a particular order. The ordered series of mutations constitutes an obligatory evolutionary pathway for the acquisition of a new function for ebgo enzyme. A comparison of newly evolved strains with parental strains shows that when ebg enzyme acquires a new function, its old functions often suffer; but that in several cases old functions are either unaffected or are improved. I conclude that divergence of functions catalyzed by an enzyme need not require gene duplication.     

A third beta-galactosidase in a strain of Klebsiella that possesses two lac genes.

Klebsiella strain RE1544 contains two lac operons, one on the chromosome and one on a lac plasmid. A mutant of RE1544, in which the lacZ genes of both operons produce no active enzyme, was found to synthesize a beta-galactosidase that hydrolyzes ortho-nitrophenyl-beta-D-galactopyranoside but not lactose. Synthesis of this beta-galactosidase (BGase-III) is induced by lactose but not by isopropyl-1-thio-beta-D-galactopyranoside or methyl-beta-D-thiogalactopyranoside. In both the regulation of synthesis and substrate specificity, BGase-III strongly resembles the ebg0 enzyme of Escherichia coli. Nevertheless, by the criteria of immunological cross-reactivity and subunit molecular weight, BGase-III is not related to the ebg0 enzyme.     

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.     

Isolation and characterization of the newly evolved ebg beta-galactosidase of Escherichia coli K-12.

The ebg beta-galactosidase of Escherichia coli K-12 strain LC110 has been purified and characterized. Strain LC110 is a Lac+ revertant of a mutant with a deletion of the lacZ beta-galactosidase gene. Its new ebg beta-galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ beta-galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o-nitrophenyl-beta-D-galactoside as substrate, the Vmax was 11,200 nmol/min per mg of enzyme, the Km was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ beta-galactosidase of wild-type E. coli K-12 were 350,000 nmol/min per mg of enzyme, 1.3 mM, and 8,000 cal/mol. A series of sugars has been examined as competitive inhibitors of ebg beta-galactosidase. Kinetic analyses suggest that ebg beta-galactosidase has a particularly high affinity for galactosamine and gamma-galactonolactone, binds galatose more tightly than lactose, and shows a general preference for monosaccharides rather than beta-galactosides. We conclude that the ebg beta-galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2-amino sugar.     

Mutational inversion of control of the lactose operon of Escherichia coli

An unusual Lac regulatory mutant of Escherichia coli K12 has been isolated and studied by genetic, physiological and biochemical techniques. In this mutant galactosides which are inducers for the wild-type organism act as corepressors, shutting off Lac enzyme synthesis. Mapping and dominance tests indicate the mutation is in the i-gene. Both in vivo and in vitro studies demonstrate that Lac repressor is made only in the presence of certain galactosides, and that the product so produced has the normal or wild-type affinity for such galactosides, yet is immune to their normal inducing action. The unusual properties of this mutant make it an excellent tool for determining the relationship between the intracellular repressor concentration and the resulting rate of Lac enzyme synthesis.     

DNA Sequence Analysis of Artificially Evolved Ebg Enzyme and Ebg Repressor Genes

The ebg system has been used as a model to study the artificial selection of new catalytic functions of enzymes and of inducer specificities of repressors. A series of mutant enzymes with altered catalytic specificities were previously characterized biochemically as were the changes in inducer specificities of mutant, but fully functional, repressors. The wild type ebg operon has been sequenced, and the sequence differences of the mutant enzymes and repressors have been determined. We now report that, contrary to our previous understanding, ebg enzyme contains 180-kD alpha-subunits and 20-kD beta-subunits, both of which are required for full activity. Mutations that dramatically affect substrate specificity and catalytic efficiency lie in two distinct regions, both well outside of the active site region. Mutations that affect inducer specificity of the ebg repressor lie within predicted sugar binding domains. Comparisons of the ebg beta-galactosidase and repressor with homologous proteins of the Escherichia coli and Klebsiella pneumoniae lac operons, and with the galactose operon repressor, suggest that the ebg and lac operons diverged prior to the divergence of E. coli from Klebsiella. One case of a triple substitution as the consequence of a single event is reported, and the implications of that observation for mechanisms of spontaneous mutagenesis are discussed.     

Regulation of Newly Evolved Enzymes. I. Selection of a Novel Lactase Regulated by Lactose in ESCHERICHIA COLI

Thirty-four lactose-utilizing strains of E. coli were selected from a lac Z deletion strain. In 31 of these, the synthesis of the newly evolved lactase is regulated by lactose. The lactase activity in all the strains is indistinguishable from the ebg(+) activity identified by Campbell, Lengyel and Langridge (1973).     

Genetic Analysis of the Glutamate Permease in Escherichia coli K-12

The glutamate permeation system in Escherichia coli K-12 consists of three genes: gltC, gltS, and gltR. The genes gltC and gltS are very closely linked, and are located between the pyrE and tna loci, in the following order: tna, gltC, gltS, pyrE; gltR is located near the metA gene. The three glt genes constitute a regulatory system in which gltR is the regulator gene responsible for the formation of repressor, gltS is the structural gene of the glutamate permease, and gltC is most probably the operator locus. The synthesis of glutamate permease is partially repressed in wild-type K-12 strains, resulting in the inability of these strains to utilize glutamate as the sole source of carbon. Derepression due to mutation at the gltC locus enables growth on glutamate as a carbon source both at 30 C and at 42 C. Temperature-sensitive gltR mutants capable of utilizing glutamate for growth at 42 C but not at 30 C were found to be derepressed for glutamate permease when grown at 42 C and partially repressed (wild-type phenotype) upon growth at 30 C. These mutants produce an altered thermolabile repressor which can be inactivated by mild heat treatment (10 min at 44 C) in the absence of growth.     

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression.

Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.     

Isolation of super-repressor mutants in the histidine utilization system of Salmonella typhimurium.

Two super-repressor mutations in the histidine utilization (hut) operons of Salmonella typhimurium are described. Cells bearing either of these mutations have levels of hut enzymes that do not increase above the uninduced levels when growth is in the presence of either histidine or the gratuitous inducer imidazole propionate. Both mutations lie in the region of the gene for the hut repressor, hutC, and reverse mutations of both are to the constitutive (repressor-negative) rather than to the inducible (wild type) phenotype. In hybrid merodiploid strains the super-repressor mutations are dominant over either wild-type (hutC+) or repressor-negative (hutC-) alleles. Whereas both super-repressor mutations cause the uninducible synthesis of hut enzymes, the degree of repression is different. One mutation causes repression of enzyme synthesis in one of the two hut operons to a level below the basal, uninduced level of wild-type cells. The other mutation causes repression to a lesser degree than in wild-type cells, so that the hut enzymes are present at a level above the normal basal level; this partially constitutive synthesis is greater for the enzymes of one of the hut operons than for the enzymes of the other. Thus, both mutations apparently result in repressors with altered operator-binding properties, in addition to altered inducer-binding properties.     

Regulation of the Synthesis of the Lactose Repressor

Measurements of the lactose repressor over a tenfold range of cell growth rates were made on protein extracts from Escherichia coli cultures grown in media with various carbon energy sources. The concentration of lactose repressor varied with the number of genome equivalents per cell over this range in growth rates, suggesting that the number of lactose molecules within the cell is determined by the number of I gene copies present. The timing of repressor synthesis during the cell division cycle and its correlation with deoxyribonucleic acid synthesis was examined by synchronizing the cell division cycle of E. coli ED1039, in which the Lac region has been transposed from 10 to 36 min on the genetic map. Measurements of lactose repressor in the synchronized culture revealed a burst of repressor synthesis at the time of I gene duplication. The concentration of lactose repressor was found to decrease as a function of total cell protein during the division cycle until an increase in synthesis occurred, suggesting that repressor synthesis probably does not occur throughout the division cycle. A model for I gene regulation is proposed.     

Lactose metabolism involving phospho-beta-galactosidase in Klebsiella.

Klebsiella strain RE1755A is a Lac- Gal- mutant which has lost both of its lac operons, but possesses a gene specifying beta-galactosidase III, an enzyme which hydrolyzes o-nitrophenyl-beta-D-galactopyranoside but does not hydrolyze lactose. Selective pressure was applied to isolate mutants able to utilize lactose. The lactose-utilizing mutants obtained were shown to possess an unaltered beta-galactosidase III. Lactose utilization was shown to result from a pleiotropic mutation which also (i) permits galactose utilization and (ii) prevents induction of beta-galactosidase III synthesis by lactose. Evidence is presented suggesting that a phospho-beta-galactosidase enzyme is involved in lactose metabolism.     

The catalytic consequences of experimental evolution. Transition-state structure during catalysis by the evolved beta-galactosidases of Escherichia coli (ebg enzymes) changed by a single mutational event.

1. The first chemical step in the hydrolysis of galactosylpyridinium ions by the evolvant ebg enzyme is less sensitive to leaving-group acidity than in the case of the wild-type ebg enzyme, implying less glycone-aglycone-bond fission at the transition state. 2. The first chemical step in the hydrolysis of aryl galactosides by ebg enzyme is probably less sensitive to leaving-group acidity than in the case of ebg enzyme, possibly as a consequence of resulting in more effective proton donation to the leaving aglycone. 3. alpha-Deuterium kinetic isotope effects of 1.1(0) and beta-deuterium kinetic isotope effects of 1.0(0) were measured for the hydrolysis of galactosyl-enzyme intermediates derived from ebg and ebg enzymes: these effects are not compatible with reaction of the sugar ring through a 4C1-like conformation, or with an ionic glycosyl-enzyme intermediate. 4. The variation with pH of steady-state kinetic parameters for hydrolysis of p-nitrophenyl galactoside by ebg and ebg enzymes and of 3-methylphenyl beta-galactoside, 3,4-dinitrophenyl beta-galactoside and beta-galactosyl-3-bromopyridinium ion by ebg enzyme was measured. The steep, non-classical, fall in activity against p-nitrophenyl galactoside at low pH observed with ebg and ebg enzymes is not observed with ebg enzymes.     

A mutation in the essential gene gmk (encoding guanlyate kinase) generates a requirement for adenine at low temperature in Salmonella enterica

In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher Km for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.