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Fitness As a Function of Β-Galactosidase Activity In Genet. Res., Camb. (1986), 48, pp. 1-8 With 3 text-figures Printed in Great Britain Fitness as a function of /?-galactosidase activity in Escherichia coli ANTONY M. DEAN, DANIEL E. DYKHUIZEN AND DANIEL L. HARTL Department of Genetics, Washington University School of Medicine, St Louis, Missouri USA 63110-1095 (Received 12 July 1985 and in revised form 13 January 1986) Summary Chemostat cultures in which the limiting nutrient was lactose have been used to study the relative growth rate of Escherichia coli in relation to the enzyme activity of /?-galactosidase. A novel genetic procedure was employed in order to obtain amino acid substitutions within the /acZ-encoded /?-galactosidase that result in differences in enzyme activity too small to be detected by ordinary mutant screens. The cryptic substitutions were obtained as spontaneous revertants of nonsense mutations within the lacZ gene, and the enzymes differing from wild type were identified by means of polyacrylamide gel electrophoresis or thermal denaturation studies. The relation between enzyme activity and growth rate of these and other mutants supports a model of intermediary metabolism in which the flux of substrate through a metabolic pathway is represented by a concave function of the activity of any enzyme in the pathway. The consequence is that small differences in enzyme activity from wild type result in even smaller changes in fitness. 1. Introduction sidase activity and growth rate in E. coli, together with a novel genetic technique for obtaining amino acid In the seminal paper, Kacser & Burns (1973) demon- strated that the net flux through a simple linear meta- substitutions that result in electrophoretic or thermo- bolic pathway should be a concave function of the lability differences from wild type. activity of a single enzyme. Subsequent experimental and theoretical work by Kacser & Burns (1979, 1981) and others (Flint et al. 1981; Middleton & Kacser, 2. Materials and Methods 1983; Heinrich & Rapoport, 1983; Kacser & Beeby, Genetic manipulations. E. coli strain CSH10 (Miller, 1984) has shown this result to be of wide application 1972) contains a nonsense mutation within the lacZ and profound importance. For example, the model has structural gene of /?-galactosidase in the lactose operon provided a compelling explanation of the recessive that abolishes enzyme activity and growth on lactose. nature of inborn errors of metabolism at the This strain was streaked out to yield single white molecular level (Kacser & Burns, 1981). colonies on MacConkey lactose indicator plates. After Within this general framework for considering and being left at 30 °C for one week, 21 red lactose- analysing complex metabolic systems comes hope for fermenting papillae were isolated from separate the elucidation of the relations between the kinetic colonies. Using PI (cm/ clrlOO), eight of these putative behaviour of enzymes, the fluxes through the path- mutants were transduced into strain DD320 ways, and the effects upon fitness. Already, some ten- (Dykhuizen & Davies, 1980), which carries a deletion tative inroads have been made. Kacser & Beeby (1984) covering the entire lac operon. This procedure ensures have considered evolution in primitive cells, Heinrich a uniform genetic background and screens out & Rapoport (1983) have suggested an approach to possible tRNA suppressors (no tRNA maps within understanding the design of metabolic systems, two minutes of the lac operon). Middleton & Kacser (1983) have studied the relation Only a small amount of /?-galactosidase activity is between the activity of alcohol dehydrogenase and required for successful PI transduction. A second /?- ethanol tolerance in Drosophila, and we have ex- galactosidase, designated evolved /?-galactosidase and plored some of the implications of the theory in the coded by the ebgA gene, has been successfully trans- context of the neutral theory of molecular evolution duced despite having a ^-galactosidase activity less (Hartl, Dykhuizen & Dean, 1985). than 0-5% of the fully induced wild-type enzyme (Hall, Here we report on the relation between /?-galacto- 1984). Therefore, a lacZ mutant resulting in an enzyme GRH48 Downloaded from https://www.cambridge.org/core. IP address: 170.106.202.8, on 29 Sep 2021 at 05:26:52, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672300024587 A. M. Dean, D. E. Dykhuizen andD. L. Hard Table 1. Origin of fi-galactosidase mutants Kinetics. The kinetic properties of the enzyme variants were studied by means of the hydrolysis of Strain Description Source o-nitrophenyl-/?-D-galactopyranoside (ONPG) in Z-buffer, conducted at 37 °C with unfractionated Inducible lac operons extracts. The concentrations of cell protein in all TDI Control CSH64 (Miller, 1974) TD10.1 Revertant CSH10 (Miller, 1974) extracts were estimated by the method of Bradford TD1O.2 Revertant CSH10 (Miller, 1974) (1976). TD10.3 Revertant CSH10 (Miller, 1974) The hydrolysis of lactose by the mutants was also TD10.4 Revertant CSH10 (Miller, 1974) studied, because ONPG is not the natural substrate of Constitutive lac operons TD2 Control JL3300 (Langridge, 1974) the enzyme. For these studies, unfractionated extracts TD3 Mutant JL1190 (Langridge, 1974) were as described earlier but with 1 % succinate (w/v) TD4 Mutant JL1481 (Langridge, 1974) as a carbon source. These were carefully sonicated in Z-buffer, in both the presence and the absence of /?-mercaptoethanol. No difference in activity per mg with greater than 0-5% wild-type activity would also protein was detected (as judged by the hydrolysis of be expected to be transduced. ONPG) and the experiments were pursued in its Two constitutive /?-galactosidase mutations, JL1190 absence. The concentrations of lactose used were from and JL1481 (Langridge, 1974), having substantially 12 mM to 0- 5 mM, except for TD4 for which the concen- reduced enzyme activity, were transduced into the trations were twenty-fold higher. DD320 background to yield strains TD3 and TD4, The production of glucose at 37 °C was monitored respectively. As controls, the wild-type lac operon of by taking samples of a reaction mix (containing less CSH64 (Miller, 1974) and the constitutive isogenic than 1 ftg/ml of cell protein) every 5 min, boiling them operon of JL3300 were also transduced into DD320 to for 3 min to stop the reaction and estimating theconcen- yield strains TDl and TD2, respectively. Table 1 lists tration of glucose using the Glucose No. 15-UV diag- these and other strains used in this research. nostic kit (Sigma Chemical Company). The kit uses Detection of substitutions. Amino acid substitutions glucose hexokinase in the presence of ATP to convert differing from wild type were detected using vertical glucose to glucose-6-phosphate, which is then oxi- polyacrylamide gel electrophoresis and thermal inacti- dized to 6-phosphogluconate by glucose-6-phosphate vation studies. Overnight cultures grown in minimal dehydrogenase in the presence of NADP. The reduc- Davis salts with 1% glucose (w/v) and 1 x 10~4 M tion of NADP to NADPH is monitored at 340 nm. isopropyl 1 -thio-/?-D-galactopyranoside (IPTG) were Every mole of NADP reduced is the equivalent of one resuspended in the same volume of Z-buffer (Miller, mole of glucose produced. With extracts from the lac 1974), sonicated and centrifuged to remove cellular deletion strain DD320, no change in absorbance was debris. In order to detect small differences in electro- detected. Although these studies demonstrated that phoretic mobility, extracts from putative variant TD3 and TD4 had less enzyme activity, those of strains and wild type (TDl) were loaded into TD10 . 3 and TD10.4 could not be distinguished from alternate lanes on several gels. Only fresh extracts were wild type (TDl). used for all protocols. Gels of 7-5% polyacrylamide The lactase activity of/?-galactosidase was also inves- with 2-5% crosslinking were pre-run for 2 h at tigated by continually monitoring the galactose pro- 15 V/cm at 12 °C in a continuous buffer of 0-1 M Tris- duced by means of a coupled assay involving the borate (pH 8.5). Samples were loaded in 40% sucrose, production of NADH from the action of galactose and electrophoresis was continued for 6 additional dehydrogenase. The same lactose concentrations as hours. The gels were then incubated at 37 °C for 1 h in mentioned earlier were used in the presence of phosphate-citrate buffer (pH 4.95) containing 0-2 g/1 lOmMNAD and 10^g/ml of galactose dehydro- of 6-bromo-2-naphthyl-/?-D-galactopyranoside, genase. The production of NADH was measured at washed with distilled water, and stained with a 1 g/1 340 nm. Estimates of Kmax were made by assuming the solution of tetrazotized-0-dianisidine until dark blue equimolar production of galactose and NADH. The bands appeared. concentrations of cell protein in the reaction mixes Heat inactivation studies were carried out at 58 °C. were between 0-1 fig/m\ and 003 /ig/ml. Samples were assayed as described by Miller (1974). As the Lineweaver-Burke plot is known to be quite About one-half of the total enzyme activity of the unreliable (Dowd & Riggs, 1964), the Kms were esti- /?-galactosidase of the wild-type strain (TDl) was lost mated using the Eadie-Hofstee plot of velocity in 1 h, whereas the heat-sensitive variants lost most of against ratio of velocity to substrate concentration. their enzyme activity after 45 min. This marked differ- The negative value of the slope of this plot yields a less ence in thermolability was readily reproducible in un- biased estimate of Km, and because both axes contain fractionated extracts. Net protein concentration had a common variable (velocity), any error will enlarge little effect on thermolability in these studies because the variance. Therefore, the tests of statistical signifi- /?-galactosidase was stabilized by the presence of cance are conservative. Estimates of Vmax were /?-mercaptoethanol in Z-buffer (Miller, 1974).
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