Specific Deletion Occurring in the Directed Evolution of 6-Phosphogluconate Dehydrogenase in ESCHERICHIA COLI

A novel genetic change leading to increased activity of 6-phosphogluconate dehydrogenase (6PGD) in E. coli has been observed. The mutation is a deletion of approximately 0.4 kilobase pairs occurring between the structural gene of 6PGD (gnd) and one copy of an insertion element (IS5) found normally in E. coli K12 a few hundred base pairs upstream (counterclockwise) from gnd at 44 minutes on the conventional genetic map. The deletion is associated with a threefold higher activity of 6PGD and a 57% increase in the maximum growth rate when cells are grown in gluconate.     

Acquisition of the rfb-gnd cluster in evolution of Escherichia coli O55 and O157

The rfb region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The rfb locus is adjacent to gnd, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if rfb and gnd cotransfer, we sequenced gnd in five O55 and 13 O157 strains of Escherichia coli. E. coli O157:H7 has a gnd allele (allele A) that is only 82% identical to the gnd allele (allele D) of closely related E. coli O55:H7. In contrast, gnd alleles of E. coli O55 in distant lineages are >99.9% identical to gnd allele D. Though gnd alleles B and C in E. coli O157 that are distantly related to E. coli O157:H7 are more similar to allele A than to allele D, there are nucleotide differences at 4 to 6% of their sites. Alleles B and C can be found in E. coli O157 in different lineages, but we have found allele A only in E. coli O157 belonging to the DEC5 lineage. DNA 3' to the O55 gnd allele in diverse E. coli lineages has sequences homologous to tnpA of the Salmonella enterica serovar Typhimurium IS200 element, E. coli Rhs elements (including an H-rpt gene), and portions of the O111 and O157 rfb regions. We conclude that rfb and gnd cotransferred into E. coli O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic E. coli O55 and O157.     

Phenotypic suppression of a fructose-1,6-diphosphate aldolase mutation in Escherichia coli

Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphosphate (FDP) aldolase; its growth on carbohydrate substrates is inhibited probably as a consequence of the accumulation of high intracellular levels of FDP. Studies of one class of phenotypic revertants of strain NP 315 which have regained their ability to grow on C(6) substrates at 40 C showed that in these strains the buildup of the inhibitory FDP pool is prevented by additional mutations in enzymes catalyzing the conversion of the substrate offered in the medium to FDP. For example, mutations affecting 6-phosphogluconate dehydrogenase activity (gnd(-)) may be selected in great number without any mutagenesis and enrichment simply by isolating revertants of strain NP 315 able to grow on gluconate at 40 C. Similarly, an additional mutation in phosphoglucose isomerase (pgi(-)) restores the ability of these fda(-)gnd(-) strains to grow on glucose at 40 C. Glucose metabolism of these fda(-)gnd(-)pgi(-) strains was investigated. The enzymes of the Entner-Doudoroff pathway are induced to an appreciable extent upon growth of these mutants on glucose medium; further evidence for glucose degradation via this route (which normally is induced only in the presence of gluconate) was provided by following the fate of the C1 label of radioactive glucose in l-alanine. Predominant labeling of the carboxyl-carbon of l-alanine was observed, inciating a major contribution of the Entner-Doudoroff path to pyruvate formation from glucose. Chromatographic analysis of the intermediates of glucose metabolism showed further that glucose apparently is at least partly metabolized via a bypass consisting of the accumulation of extracellular gluconic acid which arises by dephosphorylation of 6-phosphogluconolactone and possibly of 6-phosphogluconate. This extracellular gluconate is then taken up and metabolized in the normal manner via the Entner-Doudoroff enzymes.     

Utilization of gluconate by Escherichia coli. Uptake of d-gluconate by a mutant impaired in gluconate kinase activity and by membrane vesicles derived therefrom

1. From Escherichia coli strain K2.1.5(c).8.9, which is devoid of 6-phosphogluconate dehydrogenase (gnd) and 6-phosphogluconate dehydratase (edd) activities, a mutant R6 was isolated that was tolerant to gluconate though still edd(-), gnd(-). 2. Measurements of the fate of labelled gluconate, of the conversion of gluconate into 6-phosphogluconate, and of the induction of gluconate kinase by the two organisms show that, although both inducibly form a gluconate-transport system, strain R6 is impaired in its ability to convert the gluconate thus taken up into 6-phosphogluconate; it was therefore used for study of the kinetics and energetics of gluconate uptake. 3. Suspensions of strain R6 induced for gluconate uptake took up this substrate via a ;high affinity' transport process, with K(m) about 10mum and V(max.) about 25nmol/min per mg dry mass; a ;low affinity' system demonstrated to occur in certain E. coli mutants was not induced under the conditions used in this work. 4. The uptake of gluconate was inhibited by lack of oxygen and by inhibitors of electron transport; such inhibitors also promoted the efflux of gluconate taken up. 5. Membrane vesicles prepared from strain R6 also manifested these properties when incubated with suitable electron donors, at rates similar to those observed with whole cells. 6. The results indicate that the active transport of gluconate into the cells is the rate-limiting step in gluconate utilization by E. coli, and that the mechanism of this process can be validly studied with membrane vesicles.     

Isolation of Specialized Transducing Bacteriophages for Gluconate 6-Phosphate Dehydrogenase (gnd) of Escherichia coli

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.     

Metabolic engineering of pentose phosphate pathway in Ralstoniaeutropha for enhanced biosynthesis of poly-beta-hydroxybutyrate

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Ralstonia eutropha from gluconate as a carbon source is carried out through the Entner-Doudoroff (ED) pathway and the pentose-phosphate (PP) pathway generating NADPH and glyceraldehyde-3-phosphate that flows to acetyl-CoA, actively in the unbalanced PHB accumulation phase. The gnd gene encoding 6-phosphogluconate dehydrogenase (6PGDH) and the tktA gene encoding the transketolase (TK) in PP pathway of E. coli were transformed into R. eutropha H16 to modify the metabolic flux of gluconate to the PHB biosynthesis. Over-generated NADPH by the amplified gnd gene tended to depress the cell growth and PHB concentration. Meanwhile, the amplified tktA gene significantly increased both PHB biosynthesis and cell growth as a result of the effective flow of glyceraldehyde-3-phosphate into acetyl-CoA along with the concomitant supplementation of NADPH. The amplified tktA gene also activated the enzyme activities directly associated with PHB biosynthesis. The transformant R. eutropha harboring tktA gene was cultivated using pH-stat-fed-batch to achieve the overproduction of PHB.     

Altered growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level in hisT mutants of Salmonella typhimurium and Escherichia coli.

In Escherichia coli, the level of 6-phosphogluconate dehydrogenase is directly proportional to the cellular growth rate during growth in minimal media. This contrasts with the report by Winkler et al. (M. E. Winkler, J. R. Roth, and P. E. Hartman, J. Bacteriol. 133:830-843, 1978) that the level of the enzyme in Salmonella typhimurium LT-2 strain SB3436 is invariant. The basis for the difference in the growth-rate-dependent regulation between the two genera was investigated. Expression of gnd, which encodes 6-phosphogluconate dehydrogenase, was growth rate uninducible in strain SB3436, as reported previously, but it was 1.4-fold growth rate inducible in other S. typhimurium LT-2 strains, e.g., SA535. Both the SB3436 and SA535 gnd genes were growth rate inducible in E. coli K-12. Moreover, the nucleotide sequences of the regulatory regions of the two S. typhimurium genes were identical. We concluded that a mutation unlinked to gnd is responsible for the altered growth rate inducibility of 6-phosphogluconate dehydrogenase in strain SB3436. Transductional analysis showed that the altered regulation is due to the presence of a mutation in hisT, the gene for the tRNA modification enzyme pseudouridine synthetase I. A complementation test showed that the regulatory defect conferred by the hisT mutation was recessive. In E. coli, hisT mutations reduced the extent of growth rate induction by the same factor as in S. typhimurium. The altered regulation conferred by hisT mutations was not simply due to their general effect of reducing the polypeptide chain elongation rate, because miaA mutants, which lack another tRNA modification and have a similarity reduced chain growth rate, had higher rather than lower 6-phosphogluconate dehydrogenase levels. Studies with genetic fusions suggested that hisT mutations lower the gnd mRNA level. The data also indicated that hisT is involved in translational control of gnd expression, but not the aspect mediated by the internal complementary sequence.     

Evolution of Transposons: Natural Selection for Tn5 in ESCHERICHIA COLI K12

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.     

Growth-rate-dependent expression and cloning of gnd alleles from natural isolates of Escherichia coli.

6-Phosphogluconate dehydrogenase (6PGD), encoded by gnd, is highly polymorphic among isolates of Escherichia coli form natural populations. As a means of characterizing the growth-rate-dependent regulation of the level of 6PGD, five gnd alleles, including the E. coli B/r allele, were crossed into E. coli K-12 with bacteriophage P1. In each of the isogenic strains, the level of 6PGD was two- to threefold higher in cells grown on glucose than in cells grown on acetate. The level of enzyme activity in the acetate-grown cells varied about sixfold within the set of isogenic strains. The physiological importance of these differences in enzyme level is discussed. The gnd gene was cloned from five E. coli strains and Salmonella typhimurium LT-2 and mapped with twelve restriction endonucleases. gnd was located and oriented on the chromosomal DNAs. The restriction maps of the genes were aligned at conserved restriction sites, and the relative divergence of the genes was estimated from restriction site polymorphisms. The E. coli gnd genes differed from the S. typhimurium gene by about 11%. Most of the E. coli genes differed from one another by less than 5%, but one allele differed from the others by about 10%. Only the gnd gene from E. coli K-12 had an IS5 element located nearby.     

Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress.

The level of LamB protein in the outer membrane of Escherichia coli was derepressed in the absence of a known inducer (maltodextrins) under carbohydrate-limiting conditions in chemostats. LamB protein contributed to the ability of the bacteria to remove sugar from glucose-limited chemostats, and well-characterized lamB mutants with reduced stability constants for glucose were less growth competitive under glucose limitation than those with wild-type affinity. In turn, wild-type bacteria were less growth competitive than lamB mutants with enhanced sugar affinity. In contrast to an earlier report, we found that LamB- bacteria were less able to compete in carbohydrate-limited chemostats (with glucose, lactose, arabinose, or glycerol as the carbon and energy sources) when mixed with LamB+ bacteria. The transport Km for [14C]glucose was affected by the presence or affinity of LamB, but only in chemostat-grown bacteria, with their elevated LamB levels. The pattern of expression of LamB and the advantage it confers for growth on low concentrations of carbohydrates are consistent with a wider role in sugar permeation than simply maltosaccharide transport, and hence the well-known maltoporin activity of LamB is but one facet of its role as the general glycoporin of E. coli. A corollary of these findings is that OmpF/OmpC porins, present at high levels in carbon-limited bacteria, do not provide sufficient permeability to sugars or even glycerol to support high growth rates at low concentrations. Hence, the sugar-binding site of LamB protein is an important contributor to the permeability of the outer membrane to carbohydrates in habitats with low extracellular nutrient concentrations.     

Mathematical investigations of growth of microorganisms in the gradostat

Microbial ecosystems with spatial distribution of substrate (nutrient) supply in the form of gradients have been studied in a laboratory system called the gradostat, which is a series of coupled chemostats. We investigate analytically and numerically a mathematical model, similar to the one for a single chemostat based on Michaelis-Menten kinetics, of the growth of one species of microorganisms in the gradostat in the presence of one limiting substrate and two limiting complementary substrates. Our analysis predicts various patterns of spatial distribution of microorganisms at steady state and suggests further experiments to be performed with the gradostat.This work was supported by the Deutsche Forschungsgemeinschaft     

Regulation of Newly Evolved Enzymes. III Evolution of the ebg Repressor during Selection for Enhanced Lactase Activity

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.     

Stability and stabilization for models of chemostats with multiple limiting substrates

We study chemostat models in which multiple species compete for two or more limiting nutrients. First, we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given as positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for chemostats with one limiting nutrient. We demonstrate our methods in simulations.     

Mapping of insertion mutations in gnd of Escherichia coli with deletions defining the ends of the gene.

A genetic map was prepared for gnd, the gene of Escherichia coli which encodes the metabolically regulated 6-phosphogluconate dehydrogenase. Direct selection methods were used for the isolation of mutants with deletions that define the respective ends of gnd. These selections depended on the availability of a defective lysogen in which gnd was present on a lambda h80 dgnd his prophage located at the att phi 80 region of the chromosome. Mutants with deletions entering gnd from the his-distal end were selected as Gnd- TonB- mutants. Mutants with his-proximal gnd deletions were selected as Gnd-, temperature-resistant mutants of a specially prepared stable lysogen. Gnd- mutants were also isolated after mutagenesis with bacteriophage Mu cts61, and genetic tests were used to determine which mutants carry a Mu cts61 prophage in gnd. The deletion mutations were mapped against each other and against the insertion mutations through the use of F' merodiploid strains. The insertion mutations mapped at seven distinct sites in gnd; three mapped under the deletions defining the his-proximal portion of the gene and three mapped with the his-distal deletions.     

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.^1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression^3-6 and other characteristics of cultures at steady-state equilibrium.⁷ Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.⁸ A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.^9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. We present here the design and operation of a relatively simple, low cost array of miniature chemostats—or ministats—and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.     

Mutations affecting gluconate catabolism in Escherichia coli. Genetic mapping of the locus for the thermosensitive gluconokinase

An Escherichia coli strain unable to use gluconate was isolated by spontaneous curing of lambda cI857 s7 xis6 b515 b519, lambda cI857 s7 delta(A-att) dargI valS lysogens. Two lesions, linked to asd and pyrB markers, respectively, were necessary to produce this phenotype. The asd-linked mutation gnt-17, of regulatory type, seems to affect the expression of the major system of gluconate utilization (min 75) as well as that of 6-phosphogluconate dehydratase (gene edd, min 41), the first enzyme of the Entner-Doudoroff pathway. A closely linked suppressor of gnt-17 causes constitutivity of these activities; this suppressor resembles gntR, which is also in the asd region. Hence, it is possible that gnt-17 is a super-repressing allele of gntR, rather than a positive controlling element. Lesion gnt-17 alone does not prevent the utilization of gluconate; for this, the mutation gnt-18 at 96.9 min is also necessary. This mutation abolishes the thermosensitive gluconokinase activity and thus eliminates the subsidiary ability to catabolize gluconate. Accordingly, gnt-18 seems to be allelic with gntV, the locus postulated as being in the pyrB region specifying the thermosensitive gluconokinase.