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Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

ABSTRACT: We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of "codrugs" that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's "intrinsic resistome," which provides innate resistance to antibiotics.

SUBMITTER: Tamae C

PROVIDER: S-EPMC2519525 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

Tamae Cindy C Liu Anne A Kim Katherine K Sitz Daniel D Hong Jeeyoon J Becket Elinne E Bui Ann A Solaimani Parrisa P Tran Katherine P KP Yang Hanjing H Miller Jeffrey H JH

Journal of bacteriology 20080711 17

We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the ...[more]

PMID: 18621901

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Project description:BackgroundIt is quite important to simulate the metabolic changes of a cell in response to the change in culture environment and/or specific gene knockouts particularly for the purpose of application in industry. If this could be done, the cell design can be made without conducting exhaustive experiments, and one can screen out the promising candidates, proceeded by experimental verification of a select few of particular interest. Although several models have so far been proposed, most of them focus on the specific metabolic pathways. It is preferred to model the whole of the main metabolic pathways in Escherichia coli, allowing for the estimation of energy generation and cell synthesis, based on intracellular fluxes and that may be used to characterize phenotypic growth.ResultsIn the present study, we considered the simulation of the main metabolic pathways such as glycolysis, TCA cycle, pentose phosphate (PP) pathway, and the anapleorotic pathways using enzymatic reaction models of E. coli. Once intracellular fluxes were computed by this model, the specific ATP production rate, the specific CO? production rate, and the specific NADPH production rate could be estimated. The specific ATP production rate thus computed was used for the estimation of the specific growth rate. The CO? production rate could be used to estimate cell yield, and the specific NADPH production rate could be used to determine the flux of the oxidative PP pathway. The batch and continuous cultivations were simulated where the changing patterns of extracellular and intra-cellular metabolite concentrations were compared with experimental data. Moreover, the effects of the knockout of such pathways as Ppc, Pck and Pyk on the metabolism were simulated. It was shown to be difficult for the cell to grow in Ppc mutant due to low concentration of OAA, while Pck mutant does not necessarily show this phenomenon. The slower growth rate of the Ppc mutant was properly estimated by taking into account the lower specific ATP production rate. In the case of Pyk mutant, the enzyme level regulation was made clear such that Pyk knockout caused PEP concentration to be up-regulated and activated Ppc, which caused the increase in MAL concentration and backed up reduced PYR through Mez, resulting in the phenotypic growth characteristics similar to the wild type.ConclusionsIt was shown to be useful to simulate the main metabolism of E. coli for understanding metabolic changes inside the cell in response to specific pathway gene knockouts, considering the whole main metabolic pathways. The comparison of the simulation result with the experimental data indicates that the present model could simulate the effect of the specific gene knockouts to the changes in the metabolisms to some extent.

Project description:Metabolic networks are characterized by multiple redundant reactions that do not have a clear biological function. The redundancies in the metabolic networks are implicated in adaptation to random mutations and survival under different environmental conditions. Reactions that are not active under wild-type growth conditions, but get transiently activated after a mutation event such as gene deletion are known as latent reactions. Characterization of multiple-gene knockout mutants can identify the physiological roles of latent reactions. In this study, we characterized double-gene deletion mutants of E. coli with the aim of investigating the sub-optimal physiology of the mutants and the possible roles of latent reactions. Specifically, we investigated the effects of the deletion of the glyoxylate-shunt gene aceA (encoding a latent reaction enzyme, isocitrate lyase) on the growth characteristics of the mutant E. coli ?pgi. The deletion of aceA reduced the growth rate of E. coli ?pgi, indicating that the activation of the glyoxylate shunt plays an important role in adaptation of the mutant E. coli ?pgi when no other latent reactions are concurrently inactivated. We also investigated the effect of the order of the gene deletions on the growth rates and substrate uptake rates of the double-gene deletion mutants. The results indicate that the order in which genes are deleted determines the phenotype of the mutants during the sub-optimal growth phase. To elucidate the mechanism behind the difference between the observed phenotypes, we carried out transcriptomic analysis and constraint-based modeling of the mutants. Transcriptomic analysis showed differential expression of the gene aceK (encoding the protein isocitrate dehydrogenase kinase) involved in controlling the isocitrate flux through the TCA cycle and the glyoxylate shunt. Higher acetate production in the E. coli ?aceA1 ?pgi2 mutant was consistent with the increased aceK expression, which limits the TCA cycle flux and causes acetate production via overflow metabolism.

Dataset Information

Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

Publications

Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli.

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