Suchergebnisse

This is the final version. Available on open access from the Royal Society of Chemistry via the DOI in this record ; Data availability: All the data is available in the main text or in the supplementary materials. ; The code associated with this article is located in ORE at: http://hdl.handle.net/10871/121661 ; The double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to rapidly quantify antibiotic accumulation in hundreds of individual Escherichia coli cells. By serially manipulating the microfluidic environment, we demonstrated that stationary phase Escherichia coli, traditionally more refractory to antibiotics than growing cells, display reduced accumulation of the antibiotic ofloxacin compared to actively growing cells. Our novel microfluidic method facilitates the quantitative comparison of the role of the microenvironment versus various membrane transport pathways in cellular drug accumulation. Unlike traditional techniques, our assay is rapid, studying accumulation as the cells are dosed with the drug. This platform provides a powerful new tool for studying antibiotic accumulation in bacteria, which will be critical for the rational development of the next generation of antibiotics. ; European Commission ; Biotechnology and Biological Sciences Research Council (BBSRC) ; Engineering and Physical Sciences Research Council (EPSRC) ; University of Exeter School of Biosciences ; European Union Horizon 2020 ; Medical Research Council (MRC) ; Royal Society ; Wellcome Trust ; GW4 Initiator award

This is the final version. Available on open access from Nature Research via the DOI in this record ; Data availability; All RNA sequencing data and proteomics data is available in Supplementary Data 1–4. Exact p values, where shown on Figs. 1–5, are available in Supplementary Data 5. The source data underlying Figs. 1–5 are provided as Supplementary Data 6. Any other relevant data are available upon reasonable request. ; The interaction between a cell and its environment shapes fundamental intracellular processes such as cellular metabolism. In most cases growth rate is treated as a proximal metric for understanding the cellular metabolic status. However, changes in growth rate might not reflect metabolic variations in individuals responding to environmental fluctuations. Here we use single-cell microfluidics-microscopy combined with transcriptomics, proteomics and mathematical modelling to quantify the accumulation of glucose within Escherichia coli cells. In contrast to the current consensus, we reveal that environmental conditions which are comparatively unfavourable for growth, where both nutrients and salinity are depleted, increase glucose accumulation rates in individual bacteria and population subsets. We find that these changes in metabolic function are underpinned by variations at the translational and posttranslational level but not at the transcriptional level and are not dictated by changes in cell size. The metabolic response-characteristics identified greatly advance our fundamental understanding of the interactions between bacteria and their environment and have important ramifications when investigating cellular processes where salinity plays an important role. ; Biotechnology & Biological Sciences Research Council (BBSRC) ; Biotechnology and Biological Sciences Research Council (BBSRC) ; Medical Research Council (MRC) ; Royal Society ; QUEX Initiator grant ; European Union Horizon 2020 ; Gordon and Betty and Gordon Moore Foundation ; Wellcome Trust