Biology subjects

Overton, T. W.

Publications and source records attributed to Overton, T. W..

3 recordsLinked to original sources

bioRxiv2025

Periplasmic production of Green Fluorescent Protein is poorly tolerated by Escherichia coli

Escherichia coli is a commonly used host for recombinant protein production. It is advantageous to direct many recombinant proteins, especially those that require disulphide bonding for function, such as antibody fragments, to the periplasm of E. coli. This requires N-terminal fusion of a signal peptide that directs the polypeptide chain through the relevant translocation apparatus. Signal peptides cannot be selected on the basis of recombinant gene sequence, so screening is required to select the optimal signal peptide for each product, typically using subcellular fractionation, a time-intensive process. Fusion of a fluorescent protein such as GFP to the C-terminal of recombinant proteins has previously been used to accelerate cytoplasmic protein production process development, but most GFP proteins are not active in the periplasm. Previous studies have developed GFP derivatives that fold rapidly (such as superfolder GFP, sfGFP) and have been reported to be periplasmically active. Here, we tested the applicability of sfGFP as a periplasmic screening tool using single-cell analysis and structured illumination microscopy. We discovered that sfGFP is very poorly tolerated in the periplasm, causing deleterious effects on E. coli physiology, manifesting as poor growth, cell death, and loss of recombinant protein productivity. A further reason for poor GFP functionality in the periplasm is errant disulphide bonding, so we tested a cysteine-free GFP, which cannot form disulphide bonds; results were similar to sfGFP. In conclusion, currently-available GFP variants are poor fusion partners for screening production and translocation of recombinant proteins to the E. coli periplasm due to their negative impact on physiology. HighlightsO_LIGFP is a useful screening tool recombinant protein production. C_LIO_LIWe tested periplasmic expression of GFP derivatives sfGFP and cfSGFP2. C_LIO_LIWe used structured illumination microscopy to visualise GFP accumulation. C_LIO_LIPeriplasmic GFP derivatives have significant negative effects on bacterial physiology C_LI

bioengineering↗

bioRxiv2023

Efflux pumps mediate changes to fundamental bacterial physiology via membrane potential

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary phase cells having a re-modelled, less permeable, envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild type strain SL1344, and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarisation. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella, with likely impacts on multiple phenotypes. ImportanceWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarisation could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants.

microbiology↗

bioRxiv2020

Nanoparticle Tracking Analysis: A powerful tool for characterizing magnetosome preparations

Nanoparticle Tracking Analysis (NTA) has been employed to measure the particle concentration and size distribution of magnetosomes extracted and purified from Magnetospirillum gryphiswaldense MSR-1, and then exposed to probe ultrasonication for various times, or 1% (w/v) sodium dodecyl sulphate (SDS) for 1 h. Particle concentration increased 3.7-fold over the first 15 min of ultrasonication (from 2 × 108 to >7.3 × 108 particles mL−1), but fell steeply to ~3.6 × 108 particles mL−1 after 20 min. NTA of untreated magnetosome preparation confirmed a wide particle distribution dominated by larger species (D[1,0] = 312 nm; Dn50 = 261 nm; mode = 243 nm) with no particles in the size range of isolated single magnetosomes. After 5 min of ultrasonication the whole particle size distribution shifted to smaller size (D[1,0] = 133 nm; Dn50 = 99 nm; mode = 36 nm, corresponding to individual magnetosomes), but longer treatment times (15 and 20 min) reversed the previous transition; all characteristic numbers of the particle size distributions increased and very few small particles were detected. Side-by-side comparison of NTA and TEM sizing data revealed remarkable similarity at low ultrasonication times, with both showing single magnetosomes accounted for ~30% population after 5 min. Exposure of magnetosomes to SDS resulted in a ~3-fold increase in particle concentration to 5.8 × 108 particles mL−1, narrowing of the size distribution and gross elimination of particles below 60 nm. We conclude that NTA is a rapid cost-effective technique for measuring particle number, size distribution and aggregation state of magnetosomes in solution, but requires further work to improve its resolving power.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗