The transient kinetics of uptake of galactosides into Escherichia coli.
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ABSTRACT:
SUBMITTER: Page MG
PROVIDER: S-EPMC1144356 | biostudies-other | 1984 Nov
REPOSITORIES: biostudies-other
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Project description:1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme-substrate complex.
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Project description:Escherichia coli has homologues of the competence genes other species use for DNA uptake and processing, but natural competence and transformation have never been detected. Although we previously showed that these genes are induced by the competence regulator Sxy as in other gamma-proteobacteria, no conditions are known that naturally induce sxy expression. We have now tested whether the competence gene homologues encode a functional DNA uptake machinery and whether DNA uptake leads to recombination, by investigating the effects of plasmid-borne sxy expression on natural competence in a wide variety of E. coli strains. High- and low-level sxy expression alone did not induce transformation in any of the strains tested, despite varying the transforming DNA, its concentration, and the incubation conditions used. Direct measurements of uptake of radiolabelled DNA were below the limit of detection, however transformants were readily detected when recombination functions were provided by the lambda Red recombinase. This is the first demonstration that E. coli sxy expression can induce natural DNA uptake and that E. coli's competence genes do encode a functional uptake machinery. However, the amount of transformation cells undergo is limited both by low levels of DNA uptake and by inefficient DNA processing/recombination.
| S-EPMC3330819 | biostudies-literature
Project description:1. The transient-state and steady-state phases of the reaction between Escherichia coli alkaline phosphatase and 4-methylumbelliferyl phosphate were investigated by using a fluorimetric stopped-flow technique. 2. At low substrate concentration (5mum) in the pH range 3.8-6.3 there was an initial rapid liberation of up to 1mole of 4-methylumbelliferone/mole of enzyme. 3. At very low substrate concentration (0.1mum) in the pH range 4.9-5.9 an initial lag in 4-methylumbelliferone production was observed, from which values for k(+1) and k(-1) could be obtained. 4. The pH profiles for the rates of phosphorylation and dephosphorylation are quite different, and it is postulated that an ionizing group which determines the conformation during the phosphorylation step is not involved in the dephosphorylation step. 5. The binding constants for substrate and P(i) are similar throughout the pH range 4-8. The ionization of substrate or P(i) appeared to have no marked effect on the binding.
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Project description:How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.
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Project description:Escherichia coli chemotaxis serves as a paradigm for the way living cells respond and adapt to changes in their environment. The chemotactic response has been characterized at the level of individual flagellar motors and in populations of swimming cells. However, it has not been previously possible to quantify accurately the adaptive response of a single, multiflagellated cell. Here, we use our recently developed optical trapping technique to characterize the swimming behavior of individual bacteria as they respond to sudden changes in the chemical environment. We follow the adaptation kinetics of E. coli to varying magnitudes of step-up and step-down changes in concentration of chemoattractant. We quantify two features of adaptation and how they vary with stimulus strength: abruptness (the degree to which return to prestimulus behavior occurs within a small number of run/tumble events) and overshoot (the degree of excessive response before the return to prestimulus behavior). We also characterize the asymmetry between step-up and step-down responses, observed at the single-cell level. Our findings provide clues to an improved understanding of chemotactic adaptation.
| S-EPMC3382544 | biostudies-literature
Project description:More than half of women will experience a urinary tract infection (UTI) with uropathogenic Escherichia coli (UPEC) causing ~80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC encode functionally redundant iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7 lacks this functional redundancy and instead encodes a sole siderophore, enterobactin. To determine if E. coli HM7 possesses unidentified iron acquisition systems, we performed RNA-sequencing under iron-limiting conditions and demonstrated the ferric citrate uptake system (fecABCDE and fecIR) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is highly enriched in UPEC isolates compared to commensal or fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake (ΔentB/ΔfecA) abrogates use of ferric citrate as an iron source and fecA provides an advantage in pooled human urine in absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host Lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI.
2021-11-06 | GSE188170 | GEO
Project description:BackgroundGlobal control influences the regulation of many individual subsystems by superimposed regulator proteins. A prominent example is the control of carbohydrate uptake systems by the transcription factor Crp in Escherichia coli. A detailed understanding of the coordination of the control of individual transporters offers possibilities to explore the potential of microorganisms e.g. in biotechnology.ResultsAn o.d.e. based mathematical model is presented that maps a physiological parameter - the specific growth rate - to the sensor of the signal transduction unit, here a component of the bacterial phosphotransferase system (PTS), namely EIIACrr. The model describes the relation between the growth rate and the degree of phosphorylation of EIIA crr for a number of carbohydrates by a distinctive response curve, that differentiates between PTS transported carbohydrates and non-PTS carbohydrates. With only a small number of kinetic parameters, the model is able to describe a broad range of experimental steady-state and dynamical conditions.ConclusionThe steady-state characteristic presented shows a relationship between the growth rate and the output of the sensor system PTS. The glycolytic flux that is measured by this sensor is a good indicator to represent the nutritional status of the cell.
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Project description:The efficient sequestration of nutrients is vital for the growth and survival of microorganisms. Some nutrients, such as CO2 and NH3, are readily diffusible across the cell membrane. The large membrane permeability of these nutrients obviates the need of transporters when the ambient level is high. When the ambient level is low, however, maintaining a high intracellular nutrient level against passive back diffusion is both challenging and costly. Here, we study the delicate management of ammonium (NH4+/NH3) sequestration by E. coli cells using microfluidic chemostats. We find that as the ambient ammonium concentration is reduced, E. coli cells first maximize their ability to assimilate the gaseous NH3 diffusing into the cytoplasm and then abruptly activate ammonium transport. The onset of transport varies under different growth conditions, but always occurring just as needed to maintain growth. Quantitative modeling of known interactions reveals an integral feedback mechanism by which this need-based uptake strategy is implemented. This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum.
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Project description:MukB is a bacterial SMC (structural maintenance of chromosome) protein that regulates the global folding of the Escherichia coli chromosome by bringing distant DNA segments together. We report that moderate overproduction of MukB may lead, depending on strain and growth conditions, to transient growth arrest. In DH5? cells, overproduction of MukB or MukBEF using pBAD expression system triggered growth arrest 2.5 h after induction. The exit from growth arrest was accompanied by the loss of the overproducing plasmid and a decline in the abundance of MukBEF. The arrested cells showed a compound gene expression profile which can be characterized by the following features: (i) a broad and deep downregulation of ribosomal proteins (up to 80-fold); (ii) downregulation of groups of genes encoding enzymes involved in nucleotide metabolism, respiration, and central metabolism; (iii) upregulation of some of the genes responsive to general stress; and (iv) degradation of the patterns of spatial correlations in the transcriptional activity of the chromosome. The transcriptional state of the MukB induced arrest is most similar to stationary cells and cells recovered from stationary phase into a nutrient deprived medium, to amino acid starved cells and to the cells shifting from glucose to acetate. The mukB++ state is dissimilar from all examined transcriptional states generated by protein overexpression with the possible exception of RpoE and RpoH overexpression. Thus, the transcription profile of MukB-arrested cells can be described as a combination of responses typical for other growth-arrested cells and those for overproducers of DNA binding proteins with a particularly deep down-regulation of ribosomal genes.
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Project description:During the early stages of cytokinesis, FtsZ protofilaments form a ring-like structure, the Z-ring, in most bacterial species. This cytoskeletal scaffold recruits downstream proteins essential for septal cell wall synthesis. Despite progress in understanding the dynamic nature of the Z-ring and its role in coordinating septal cell wall synthesis, the early stages of protofilament formation and subsequent assembly into the Z-ring are still not understood. Here we investigate a sequence of assembly steps that lead to the formation of the Z-ring in Escherichia coli using high temporal and spatial resolution imaging. Our data show that formation of the Z-ring is preceded by transient membrane-linked FtsZ assemblies. These assemblies form after attachment of short cytosolic protofilaments, which we estimate to be less than 20 monomers long, to the membrane. The attachments occur at random locations along the length of the cell. The filaments treadmill and show periods of rapid growth and shrinkage. Their dynamic properties imply that protofilaments are bundled in these assemblies. Furthermore, we establish that the size of assemblies is sensitively controlled by the availability of FtsZ molecules and by the presence of ZapA proteins. The latter has been implicated in cross-linking the protofilaments. The likely function of these dynamic FtsZ assemblies is to sample the cell surface for the proper location for the Z-ring.
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