Uptake of tetracycline by membrane preparations from Escherichia coli.
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ABSTRACT:
SUBMITTER: Franklin TJ
PROVIDER: S-EPMC1176932 | biostudies-other | 1971 Jun
REPOSITORIES: biostudies-other
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Project description:The antibiotic fosfomycin is widely recognized for treatment of lower urinary tract infections caused by Escherichia coli and lately gained importance as a therapeutic option to combat multidrug resistant bacteria. Still, resistance to fosfomycin frequently develops through mutations reducing its uptake. Whereas the inner membrane transport of fosfomycin has been extensively studied in E. coli, its outer membrane (OM) transport remains insufficiently understood. While evaluating minimal inhibitory concentrations in OM porin-deficient mutants, we observed that the E. coli ΔompCΔompF strain is five times more resistant to fosfomycin than the wild type and the respective single mutants. Continuous monitoring of cell lysis of porin-deficient strains in response to fosfomycin additionally indicated the relevance of LamB. Furthermore, the physiological relevance of OmpF, OmpC and LamB for fosfomycin uptake was confirmed by electrophysiological and transcriptional analysis. This study expands the knowledge of how fosfomycin crosses the OM of E. coli.
2024-01-24 | GSE236554 | GEO
Project description:1. At low concentrations of tetracycline (10mug/ml) net accumulation of the drug by Escherichia coli cells ceased after 7-10min. 2. At higher concentrations of tetracycline (>30mug/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10mug of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200mug of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400mug/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline, but promoted both efflux of tetracycline and cell lysis in cells treated with a high concentration of tetracycline. 8. A study of the distribution of tetracycline among the subcellular fractions of penicillin-induced spheroplasts preincubated with various concentrations of tetracycline indicated that 60-70% of the accumulated tetracycline was in the high-speed supernatant fraction. Sephadex chromatography showed that the tetracycline of this fraction was present as the free drug. Sephadex chromatography of a detergent extract of the membrane fraction, however, indicated that a significant proportion of the tetracycline radioactivity of this fraction was apparently bound to some macromolecular component. 9. Cellulose phosphate paper chromatography of cold-acid extracts of spheroplasts preloaded with tetracycline indicated that the accumulated drug was chemically unchanged. 10. Membrane preparations isolated from osmotically lysed penicillin-induced spheroplasts showed a temperature-dependent binding of tetracycline that was not energy-dependent and was not inhibited by N-ethylmaleimide. The binding process was stimulated by omitting Mg(2+) from the medium, but conversely was profoundly inhibited by EDTA. 11. The relevance of these findings to the probable mechanism of active tetracycline accumulation by E. coli is discussed.
| S-EPMC1185357 | biostudies-other
Project description:BackgroundThe production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes.ResultsA set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities.ConclusionsThe use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.
| S-EPMC3121589 | biostudies-literature
Project description:We characterized the global transcriptome of Escherichia coli MG1655:: tetA grown in the presence of ½ MIC (14?mg/L) of OTC, and for comparison WT MG1655 strain grown with 1//2 MIC of OTC (0.25?mg/L OTC). 1646 genes changed expression significantly (FDR?>?0.05) in the resistant strain, the majority of which (1246) were also regulated in WT strain. Genes involved in purine synthesis and ribosome structure and function were top-enriched among up-regulated genes, and anaerobic respiration, nitrate metabolism and aromatic amino acid biosynthesis genes among down-regulated genes. Blocking of the purine-synthesis- did not affect resistance phenotypes (MIC and growth rate with OTC), while blocking of protein synthesis using low concentrations of chloramphenicol or gentamicin, lowered MIC towards OTC. Metabolic-modeling, using a novel model for MG1655 and continuous weighing factor that reflected the degree of up or down regulation of genes encoding a reaction, identified 102 metabolic reactions with significant change in flux in MG1655:: tetA when grown in the presence of OTC compared to growth without OTC. These pathways could not have been predicted by simply analyzing functions of the up and down regulated genes, and thus this work has provided a novel method for identification of reactions which are essential in the adaptation to growth in the presence of antimicrobials.
| S-EPMC7242477 | biostudies-literature
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:Manure compost has been thought of as a potential important route of transmission of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) from livestock to humans. To clarify the abundance of ARB and ARGs, ARB and ARGs were quantitatively determined in tetracycline-resistant Escherichia coli (harboring the tetA gene)-spiked feces in simulated composts. In the simulated composts, the concentration of spiked E. coli decreased below the detection limit at day 7. The tetA gene remained in manure compost for 20 days, although the levels of the gene decreased. Next, to clarify the field conditions of manure compost in Japan, the quantities of tetracycline-resistant bacteria, tetracycline resistance genes, and residual tetracyclines were determined using field-manure-matured composts in livestock farms. Tetracycline-resistant bacteria were detected in 54.5% of tested matured compost (6/11 farms). The copy number of the tetA gene and the concentrations of residual tetracyclines in field manure compost were significantly correlated. These results suggest that the use of antimicrobials in livestock constitutes a selective pressure, not only in livestock feces but also in manure compost. The appropriate use of antimicrobials in livestock and treatment of manure compost are important for avoiding the spread of ARB and ARGs.
| S-EPMC7168215 | 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:BackgroundAntimicrobial helper-compounds may reverse antimicrobial resistance. Sertraline, a antidepressant drug, has been suggested as a tetracycline helper-compound. Tetracycline is the preferred antimicrobial for treatment of enteric diseases in pigs. This study is the first to evaluate the potency of sertraline as a tetracycline adjuvant in pigs.MethodsForty-eight nursery pigs were divided into four treatment groups: Tetracycline, sertraline, tetracycline/sertraline or un-medicated control. Fecal and ileal samples were obtained before treatment, 48?h and nine days after five days of treatment, respectively. Colony forming units (CFU) of tetracycline resistant coliforms in each sample (ileal or fecal) and CFU of an orally inoculated tetracycline-resistant strain of Escherichia coli were determined at each sampling point. The microbiome of fecal and ileal and samples was analyzed by sequencing of the 16S V3-V4 region.ResultsThe results did not provide evidence that sertraline in combination with tetracycline has any impact on tetracycline resistant bacteria in either fecal or ileum samples, while in the tetracycline treated group of pigs, an increase in the prevalence of a tetracycline resistant indicator strain of Escherichia coli shortly after ended five-day treatment was observed. The ileal samples obtained shortly after ended treatment showed treatment-associated changes in the composition of the microbiota in the groups of pigs treated with tetracycline (+/-) sertraline. While tetracycline treatment increased the abundance in the reads of E. coli, sertraline/tetracycline treatment led to increased abundances of Streptococcus spp. and decreased abundances of Lactobacillus spp. However, all observed differences (on CFU counts and microbiota composition) between groups shortly after treatment had diminished in less than two weeks after last treatment day.ConclusionsSertraline (+/-) tetracycline treatment did not reduce the long-term level of tetracycline-resistant bacteria in the feces or small intestine contents of piglets compared to the un-medicated control group of pigs. The result of this study reflects the importance of in vivo studies for confirmation of the antimicrobial helper-compound potential of an in vitro active compound.
| S-EPMC6330422 | biostudies-literature
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.
| S-EPMC2148058 | biostudies-literature
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.
| S-EPMC3472687 | biostudies-literature