Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli.
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ABSTRACT:
SUBMITTER: Lee HY
PROVIDER: S-EPMC1790958 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: Not available
| S-EPMC6494352 | biostudies-literature
Project description:Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6-deoxyerythronolide B (6-dEB) or 3-?-mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60- and 25-fold more erythromycin D (EryD) than the original strain K207-3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.
| S-EPMC3815289 | biostudies-literature
Project description:Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ?1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.
| S-EPMC4808198 | biostudies-literature
Project description:Microarrays allow us to monitor the change in transcription of every gene in the genome in response to a change in cellular state. We use cDNA microarrays to measure the response of E. coli to 13 different antibiotics and 3 synergistic combinations. Hierarchichal clustering reveals 4 distinct classes of antibiotics distinguished by their modes of action, and allows us to predict the mechanism for promethazine, a drug whose mode of action has not previously been established. The expression profiles of the synergistic combinations exhibit a complex relationship between the two component antibiotics, with similarity to one of the two drugs, as well as a surprising number of new gene responses exhibited by E. coli in response to neither drug alone. The subset of drugs which act in synergy with each other suggests that only very specific combination of mechanisms give rise to synergistic behavior. Keywords: stress response, antibiotic response, synergy
2008-02-12 | GSE10440 | GEO
Project description: Not available
| S-EPMC6812442 | biostudies-literature
Project description:Macrolides are clinically important antibiotics thought to inhibit bacterial growth by impeding the passage of newly synthesized polypeptides through the nascent peptide exit tunnel of the bacterial ribosome. Recent data challenged this view by showing that macrolide antibiotics can differentially affect synthesis of individual proteins. In order to understand the general mechanism of macrolide action, we used genome-wide ribosome profiling and analyzed the redistribution of ribosomes translating highly expressed genes in bacterial cells treated with high concentrations of macrolide antibiotics. The metagene analysis indicated that inhibition of early rounds of translation, which would be characteristic of the conventional view of macrolide action, occurs only at a limited number of genes. Translation of most genes proceeds past the 5' proximal codons and can be arrested at more distal codons when the ribosome encounters specific short sequence motifs. The sequence motifs enriched in the sites of arrest are confined to the nascent peptide residues in the peptidyl transferase center but not to the peptide segments that contact the antibiotic molecule in the exit tunnel. This led to the conclusion that the general mode of macrolide action involves selective inhibition of peptide bond formation between specific combinations of donor and acceptor substrates. Additional factors operating in the living cell but not during in vitro protein synthesis may modulate site-specific action of macrolide antibiotics.
2014-11-03 | GSE61619 | GEO
Project description:Escherichia coli has been considered as a promising host for the production of N-glycosylated therapeutic proteins and glycoconjugate vaccines. In this study, we developed a simple and efficient strategy for improving the production of N-glycosylated recombinant proteins by combining auto-induction with the use of a leaky E. coli strain. A leaky E. coli strain, designated as CLM37-Δlpp, was engineered by deleting the Braun's lipoprotein (lpp) gene of E. coli strain CLM37. Three distinct acceptor model N-glycosylated proteins, glyco-tagged human tenth fibronectin type III domain (FN3-Gly), enhanced green fluorescent protein (eGFP-Gly), and scFv of vascular endothelial growth factor receptor 3 (scFv-VEGFR3-Gly) were then expressed in CLM37-Δlpp, which carried an N-glycosylation machinery from Campylobacter jejuni for the investigation of glycoprotein production. As much as 75%, 65%, and 60% of the glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly, respectively, were found in the culture medium. The yields of glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly were 106 ± 7.4 mg/L, 65 ± 2.5 mg/L, and 62 ± 4.3 mg/L, respectively, which were more than three folds the corresponding yields obtained when these proteins were expressed in CLM37, the unmodified strain. The results suggested that this simplified approach could improve the production of N-glycosylated proteins with E. coli to facilitate large-scale production.
| S-EPMC6646630 | biostudies-literature
Project description:Urinary tract infections (UTIs) are the most common bacterial infections around the world. Uropathogenic Escherichia coli (UPEC) is among the main pathogens isolated in UTIs. The rate of UPEC with high resistance towards antibiotics and multidrug-resistant bacteria have increased dramatically and conduct to the difficulty to treat UTIs. Due to the rarefaction of new antibiotics molecules, new alternative strategies must be evaluated. Since many years, propolis has demonstrated an interesting antibacterial activity against E. coli. Here, we evaluated its activity added to antibiotics on a panel of UPEC with different resistance mechanisms. Minimal inhibitory concentrations (MICs) and time-kill curves of fosfomycin, ceftriaxone, ertapenem and ofloxacin, with and without propolis, were determined. Significant diminution of the MICs was observed using ceftriaxone or ofloxacin + propolis. Propolis alone had a bacteriostatic activity with time-dependent effect against UPEC. The addition of this nutraceutical improved the effect of all the antibiotics evaluated (except fosfomycin) and showed a synergistic bactericidal effect (fractional inhibitory concentrations index ≤ 0.5 and a decrease ≥ 2 log CFU/mL for the combination of propolis plus antibiotics compared with the antibiotic alone). Propolis is able to restore in vitro antibiotic susceptibility when added to antibiotics against UPEC. This study showed that propolis could enhance the efficiency of antibiotics used in UTIs and could represent an alternative solution.
| S-EPMC7692270 | biostudies-literature
Project description:Temperature variation-through time and across climatic gradients-affects individuals, populations, and communities. Yet how the thermal response of biological systems is altered by environmental stressors is poorly understood. Here, we quantify two key features-optimal temperature and temperature breadth-to investigate how temperature responses vary in the presence of antibiotics. We use high-throughput screening to measure growth of Escherichia coli under single and pairwise combinations of 12 antibiotics across seven temperatures that range from 22°C to 46°C. We find that antibiotic stress often results in considerable changes in the optimal temperature for growth and a narrower temperature breadth. The direction of the optimal temperature shifts can be explained by the similarities between antibiotic-induced and temperature-induced damage to the physiology of the bacterium. We also find that the effects of pairs of stressors in the temperature response can often be explained by just one antibiotic out of the pair. Our study has implications for a general understanding of how ecological systems adapt and evolve to environmental changes. IMPORTANCE The growth of living organisms varies with temperature. This dependence is described by a temperature response curve that is described by an optimal temperature where growth is maximized and a temperature range (termed breadth) across which the organism can grow. Because an organism's temperature response evolves or acclimates to its environment, it is often assumed to change over only evolutionary or developmental timescales. Counter to this, we show here that antibiotics can quickly (over hours) change the optimal growth temperature and temperature breadth for the bacterium Escherichia coli. Moreover, our results suggest a shared-damage hypothesis: when an antibiotic damages similar cellular components as hot (or cold) temperatures do, this shared damage will combine and compound to more greatly reduce growth when that antibiotic is administered at hot (or cold) temperatures. This hypothesis could potentially also explain how temperature responses are modified by stressors other than antibiotics.
| S-EPMC8422994 | biostudies-literature
Project description:BackgroundExplanations for bacterial biofilm persistence during antibiotic treatment typically depend on non-genetic mechanisms, and rarely consider the contribution of evolutionary processes.ResultsUsing Escherichia coli biofilms, we demonstrate that heritable variation for broad-spectrum antibiotic resistance can arise and accumulate rapidly during biofilm development, even in the absence of antibiotic selection.ConclusionsOur results demonstrate the rapid de novo evolution of heritable variation in antibiotic sensitivity and resistance during E. coli biofilm development. We suggest that evolutionary processes, whether genetic drift or natural selection, should be considered as a factor to explain the elevated tolerance to antibiotics typically observed in bacterial biofilms. This could be an under-appreciated mechanism that accounts why biofilm populations are, in general, highly resistant to antibiotic treatment.
| S-EPMC3568021 | biostudies-literature