Bacterial ageing in the absence of external stressors

This is the final version. Available from the Royal Society via the DOI in this record.Evidence of ageing in the bacterium Escherichia coli was a landmark finding in senescence research, as it suggested that even organisms with morphologically symmetrical fission may have evolved strategies to permit damage accumulation. However, recent work has suggested that ageing is only detectable in this organism in the presence of extrinsic stressors, such as the fluorescent proteins and strong light sources typically used to excite them. Here we combine microfluidics with brightfield microscopy to provide evidence of ageing in E. coli in the absence of these stressors. We report (i) that the doubling time of the lineage of cells that consistently inherits the 'maternal old pole' progressively increases with successive rounds of cell division until it reaches an apparent asymptote, and (ii) that the parental cell divides asymmetrically, with the old pole daughter showing a longer doubling time and slower glucose accumulation than the new pole daughter. Notably, these patterns arise without the progressive accumulation or asymmetric partitioning of observable misfolded-protein aggregates, phenomena previously hypothesized to cause the ageing phenotype. Our findings suggest that ageing is part of the naturally occurring ecologically-relevant phenotype of this bacterium and highlight the importance of alternative mechanisms of damage accumulation in this context.Medical Research CouncilEPSRCBBSRCRoyal SocietyWellcome Trus

Fluorescent macrolide probes – synthesis and use in evaluation of bacterial resistance

This is the final version. Available on open access from the Royal Society of Chemistry via the DOI in this recordThe emerging crisis of antibiotic resistance requires a multi-pronged approach in order to avert the onset of a post-antibiotic age. Studies of antibiotic uptake and localisation in live cells may inform the design of improved drugs and help develop a better understanding of bacterial resistance and persistence. To facilitate this research, we have synthesised fluorescent derivatives of the macrolide antibiotic erythromycin. These analogues exhibit a similar spectrum of antibiotic activity to the parent drug and are capable of labelling both Gram-positive and -negative bacteria for microscopy. The probes localise intracellularly, with uptake in Gram-negative bacteria dependent on the level of efflux pump activity. A plate-based assay established to quantify bacterial labelling and localisation demonstrated that the probes were taken up by both susceptible and resistant bacteria. Significant intra-strain and -species differences were observed in these preliminary studies. In order to examine uptake in real-time, the probe was used in single-cell microfluidic microscopy, revealing previously unseen heterogeneity of uptake in populations of susceptible bacteria. These studies illustrate the potential of fluorescent macrolide probes to characterise and explore drug uptake and efflux in bacteria.Australian Postgraduate AwardInstitute for Molecular Biosciences Research Advancement AwardMedical Research Council (MRC)Gordon and Betty and Gordon Moore FoundationCampus France the Programme Hubert Curien FASIC 2018Wellcome TrustNHMR

Bacteriostatic antibiotics promote CRISPR-Cas adaptive immunity by enabling increased spacer acquisition

This is the final version. Available on open access from Cell Press via the DOI in this recordData and code availability: Source data are available at Mendeley Data: https://doi.org/10.17632/gbdfwg325y.1 This paper does not report original code. Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon request.Phages impose strong selection on bacteria to evolve resistance against viral predation. Bacteria can rapidly evolve phage resistance via receptor mutation or using their CRISPR-Cas adaptive immune systems. Acquisition of CRISPR immunity relies on the insertion of a phage-derived sequence into CRISPR arrays in the bacterial genome. Using Pseudomonas aeruginosa and its phage DMS3vir as a model, we demonstrate that conditions that reduce bacterial growth rates, such as exposure to bacteriostatic antibiotics (which inhibit cell growth without killing), promote the evolution of CRISPR immunity. We demonstrate that this is due to slower phage development under these conditions, which provides more time for cells to acquire phage-derived sequences and mount an immune response. Our data reveal that the speed of phage development is a key determinant of the evolution of CRISPR immunity and suggest that use of bacteriostatic antibiotics can trigger elevated levels of CRISPR immunity in human-associated and natural environments.European Union Horizon 2020Natural Environment Research Council (NERC)Ministry of Science and Higher Education of the Russian FederationNational Institutes of Health (NIH)Russian Science Foundatio

Gradient-free determination of isoelectric points of proteins on chip

The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, $\beta$-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.We acknowledge financial support from the Biotechnology and Biological Sciences Research Council, the Newman Foundation and the Elan Pharmaceuticals. The research leading to these results has also received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007- 2013) through the ERC grant PhysProt (agreement n1 337969)

Heterologous Protein Expression Favors the Formation of Protein Aggregates in Persister and Viable but Nonculturable Bacteria

This is the final version. Available from the American Chemical Society via the DOI in this record. Environmental and intracellular stresses can perturb protein homeostasis and trigger the formation and accumulation of protein aggregates. It has been recently suggested that the level of protein aggregates accumulated in bacteria correlates with the frequency of persister and viable but nonculturable cells that transiently survive treatment with multiple antibiotics. However, these findings have often been obtained employing fluorescent reporter strains. This enforced heterologous protein expression facilitates the visualization of protein aggregates but could also trigger the formation and accumulation of protein aggregates. Using microfluidics-based single-cell microscopy and a library of green fluorescent protein reporter strains, we show that heterologous protein expression favors the formation of protein aggregates. We found that persister and viable but nonculturable bacteria surviving treatment with antibiotics are more likely to contain protein aggregates and downregulate the expression of heterologous proteins. Our data also suggest that such aggregates are more basic with respect to the rest of the cell. These findings provide evidence for a strong link between heterologous protein expression, protein aggregation, intracellular pH, and phenotypic survival to antibiotics, suggesting that antibiotic treatments against persister and viable but nonculturable cells could be developed by modulating protein aggregation and pH regulation.The Royal SocietyMarie Skłodowska‐CurieBiotechnology and Biological Sciences Research Council (BBSRC)Medical Research Council (MRC)The Gordon and Betty Moore FoundationMedical Research Council (MRC)Engineering and Physical Sciences Research Council (EPSRC)University of ExeterDST

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

This is the final version. Available on open access from Nature Research via the DOI in this record. The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.Wellcome TrustBiotechnology and Biological Sciences Research Council (BBSRC)Medical Research Council (MRC)Gordon and Betty Moore FoundationEuropean Research Council (ERC)Biotechnology and Biological Sciences Research CouncilAustralian Postgraduate Award (APA)IMB Research Advancement Awar

Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes

This is the final version. Available on open access from the Public Library of Science via the DOI in this recordData Availability: All relevant data are within the paper's Supporting Information files. Numerical values for Fig 4 can be found at https://doi.org/10.6084/m9.figshare.22086647One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 μm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.Gordon and Betty and Gordon Moore FoundationBiotechnology and Biological Sciences Research Council (BBSRC)European Union Horizon 2020Volkswagen FoundationMerton College, University of Oxford (NATI

Fast bacterial growth reduces antibiotic accumulation and efficacy

This is the final version. Available from eLife Sciences Publications via the DOI in this record. Data availability: All data acquired for this study are presented within the manuscript, the supplementary information and the source data files.Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.Engineering and Physical Sciences Research Council (EPSRC)Biotechnology & Biological Sciences Research Council (BBSRC)Medical Research CouncilGordon and Betty Moore FoundationEngineering and Physical Sciences Research CouncilWellcome TrustRoyal SocietyH2020 Marie Skłodowska-Curie Action

Synthesis of vancomycin fluorescent probes that retain antimicrobial activity, identify Gram-positive bacteria, and detect Gram-negative outer membrane damage

This is the final version. Available from Nature Research via the DOI in this record. All relevant data are available in this article, its Supplementary Information and Supplementary Data files (the source data behind the graphs in the paper is contained in Supplementary Data 2), except for original image files, which are available from the corresponding author upon reasonable request.Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics.Biotechnology & Biological Sciences Research Council (BBSRC)Engineering and Physical Sciences Research Council (EPSRC)Wellcome TrustMedical Research Council (MRC)Gordon and Betty Moore Foundation Marine Microbiology InitiativeNHMRCNHMRCNHMRCThe Royal SocietyChina Scholarship Council (CSC)University of QueenslandInstitute for Molecular BiosciencesQUEXGW

Nutrient and salt depletion synergistically boosts glucose metabolism in individual Escherichia coli cells

This is the final version. Available on open access from Nature Research via the DOI in this recordData 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 SocietyQUEX Initiator grantEuropean Union Horizon 2020Gordon and Betty and Gordon Moore FoundationWellcome Trus