Glucose transport of Escherichia coli growing in glucose-limited continuous culture.
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ABSTRACT:
SUBMITTER: Hunter IS
PROVIDER: S-EPMC1186484 | biostudies-other | 1979 Jan
REPOSITORIES: biostudies-other
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Project description:Growth of Escherichia coli K12 in a chemostat was limited by sulphate concentrations lower than 300 muM. The synthesis of extracellular polysaccharide and a change in morphology accompanied sulphate-limited growth. Growth yields with respect to the amount of glycerol or oxygen consumed were sixfold and twofold lower respectively under these conditions than when growth was limited by glycerol. Sulphate-limited cells lacked the proton-translocating oxidoreduction segment of the electron-transport chain between NADH and the cytochromes, and particles prepared from these cells lacked the energy-dependent reduction of NAD+ by succinate, DL-alpha-glycerophosphate or D-lactate, suggesting the loss of site-I phosphorylation. Glycerol-limited cells contained cytochrome b556, b562 and o, ubiquinone and low concentrations of menaquinone. Sulphate limitation resulted in the additional synthesis of cytochromes d, a1, b558 and c550; the amount of ubiquinone was decreased and menaquinone was barely detectable. Non-haem iron and acid-labile sulphide concentrations were twofold lower in electron-transport particles prepared from sulphate-limited cells. Recovery of site-I phosphorylation could not be demonstrated after incubating sulphate-limited cells with or without glycerol, in either the absence or presence of added sulphate. The loss of site-I phosphorylation in sulphate-limited cells is discussed with reference to the accompanying alterations in cytochrome composition of such cells. Schemes are proposed for the functional organization of the respiratory chains of E. coli grown under conditions of glycerol or sulphate limitation.
| S-EPMC1172507 | biostudies-other
Project description:: Bacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200-300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.
| S-EPMC3534542 | biostudies-literature
Project description:The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters ?(max) and K(s) were determined for growth with glucose. For both strains, ?(max) was determined in batch culture after different precultivation conditions. In the case of C. heintzii, ?(max) was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h(-1) C. heintzii grew immediately with a ?(max) of 0.17 ± 0.03 h(-1). After five transfers in batch culture, ?(max) had increased only slightly to 0.18 ± 0.03 h(-1). A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D = 0.075 h(-1) into glucose-excess batch medium E. coli grew only after an acceleration phase of ~3.5 h with a ?(max) of 0.52 h(-1). After 120 generations and several transfers into fresh medium, ?(max) had increased to 0.80 ± 0.03 h(-1). For long-term adapted chemostat-cultivated cells, a K(s) for glucose of 15 ?g l(-1) for C. heintzii, and of 35 ?g l(-1) for E. coli, respectively, was determined in (14)C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h(-1). Using the determined pure culture parameter values for K(s) and ?(max), it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s(min)). The values estimated for s(min) were dependent on growth rate; at D = 0.05 h(-1), it was 12.6 and 0 ?g l(-1) for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h(-1), s(min) for E. coli had to be raised to 34.9 ?g l(-1) whereas s(min) for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K(s) value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s(min).
| S-EPMC3309354 | biostudies-literature
Project description:The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.
| S-EPMC3457515 | biostudies-literature
Project description:Considerable differences in steady-state hexokinase specific activity were found in 16 N.C.I.B. strains of Klebsiella aerogenes grown in identical conditions in glucose-limited chemostats. Strains of N.C.I.B. 8258 had no detectable activity, but its glucose-phosphoenolpyruvate phosphotransferase specific activity and that of the other strains were closely similar, and it is concluded that this phosphotransferase activity regulates the overall utilization of glucose, in which hexokinase plays no essential role. The hexokinase activity was subject to regulation by the availability of phosphorus, but this did not affect the glucose phosphotransferase activity. tlactose-grown organisms (including strain N.C.I.B. 8258) had no glucose phosphotransferase activity, but more than adequate hexokinase activity to phosphorylate the intracellularly liberated glucose.
| S-EPMC1165052 | biostudies-other
Project description:A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic "work mode." sigma(S)-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.
| S-EPMC2583496 | biostudies-literature
Project description:1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH-activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (K(m) 0.25-0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO(4) (3-)-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F(-), PO(4) (3-)) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.
| S-EPMC1178562 | biostudies-other
Project description:In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (O) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not according to the doughnut model.
| S-EPMC4428220 | biostudies-literature
Project description:Transcription profile of Escherichia coli cells in biofilms under static batch culture was compared to that of E. coli cells in planktonic cultures. Both E. coli biofilm and planktonic cultures were cultivated for 18 h in 10% Luria-Bertani broth at room temperature (20 degree Celsius). Biofilms were grown in static batch culture in petri dishes. Both planktonic culture and biofilms were homogenized and run through a separated protocol.
2013-12-30 | GSE24914 | GEO
Project description:1. Pullulanase synthesis was studied in 16 classified (N.C.I.B.) strains and in an industrial strain (R) of Klebsiella aerogenes grown in chemostats containing maltose as inducer and sole carbon source. 2. Maximum synthesis was associated with carbon-limited growth at a low dilution rate (about 0.2h(-1)). The enzyme remained firmly cell-bound and seemed to be located on the cell surface. 3. Three strains had high activity (R, N.C.I.B. 5938, 8017), twelve were intermediate, and two (N.C.I.B. 8153, 9146) had negligible activity but were inducible with pullulan. 4. Pullulan similarly induced low, but adequate, activity in the other strains in conditions (nutrient limitation other than carbon-limitation) in which pullulanase was otherwise very seriously repressed. Nevertheless, in carbon limitation pullulan induced no more enzyme than did maltose, maltotriose or oligosaccharide mixtures, and ;hyperactivity' never developed on protracted culture. 5. Cyclic AMP relieved the transient repression produced by adding glucose to maltose-limited cultures and a further change to glucose-limited conditions led to constitutive pullulanase synthesis. 6. Amylomaltase and alpha-glucosidase activities were also examined but in less detail. 7. The presence of pullulanase in maltose-limited growth is discussed, but no clear function can be assigned to it at present. The molar growth yields for all the strains were very similar, and no correlation was found between the overgrowth of one strain by another and pullulanase activity. Further, any function as a general branching enzyme in polysaccharide synthesis seems unlikely.
| S-EPMC1168509 | biostudies-other