Biology subjects

Krasovec, R.

Publications and source records attributed to Krasovec, R..

9 recordsLinked to original sources

bioRxiv2025

Identifying rare spontaneous mutations through wildtype E. coli population sequencing

Understanding the rate and types of mutations occurring in different populations is fundamental to our ability to predict and potentially manipulate evolution. Common techniques for microbial mutation rate estimation fall into 2 camps; rapid, locus specific, generally liquid-culture based fluctuation assays; and slow, genome-wide, generally solid-culture based mutation accumulation experiments. This constrains the hypotheses which can be tested, notably the mutagenic effects of liquid environments cannot be rapidly quantified at a genome-wide scale. One example of such effect is the negative association previously observed between population density and mutation rates at many marker loci using the fluctuation assay. Because homogeneous population density is specifically relevant in liquid culture, this association has not been tested at a truly genome-wide scale. We fill this methodological gap by developing a novel pipeline that relies on population sequencing to capture how mutation rates are affected by population density in liquid cultures. We simulate expected mutation counts in a growing population along with random sampling during sequencing to estimate the necessary sequencing coverage as [≥]1,000-fold. We then carry out PCR-free sequencing of 95 wildtype E. coli populations at this coverage, calling rare variants with both reference-based and reference-free methods. These variant-calling methods are prone to different sources of error which can be minimised by considering only mutations called by both pipelines. This approach identifies 119 mutations across all cultures, with (non)coding/(non)synonymous and mutational spectrum profiles consistent with being samples of spontaneous mutation unbiased by selection. The distribution of these mutations also supports the motivating hypothesis, finding that mutation counts across the genome are strongly negatively associated with population density. This demonstrates the utility of population sequencing for the rapid testing of many previously inaccessible evolutionary biology hypotheses.

evolutionary biology↗

bioRxiv2025

Evolutionary potential of the Escherichia coli antimutator ΔnudJ is reduced via altered mutational spectrum

The rate of spontaneous mutation is a key factor in determining the capacity of a population to adapt to a novel environment, for example a bacterial population exposed to antibiotics. Genetic and environmental factors controlling the mutation rate commonly also cause shifts in the relative rates of different mutational classes, i.e. the spectrum of spontaneous mutations. When the mutational spectrum is altered, the relatively enriched and depleted mutations may differ in their fitness effects. Here we aim to explore how a reduced mutation rate and altered mutational spectrum can contribute to adaptation in Escherichia coli. We measure mutation rates across a set of Nudix hydrolase deletants, finding multiple strains with an antimutator phenotype. We focus on the antimutator {Delta}nudJ which can cause a 6-fold mutation rate reduction relative to the wildtype with an altered mutational spectrum. Not only does nudJ deletion reduce the probability of antibiotic resistance arising but, in the case of rifampicin, the accessible resistance mutations under this altered mutation spectrum have greater fitness costs. While this effect is, so far, specific to this drug, it opens up the possibility of reducing the likelihood of resistance mutations establishing through simultaneous modification of mutation rate and spectrum. The identification and characterisation of antimutator alleles such as {Delta}nudJ provides potential targets for antievolution drugs which could be applied during antibiotic treatment to inhibit NudJ activity. Such an approach could suppress spontaneous resistance evolution both through fewer resistance mutations and by limiting access to the fittest mutations.

evolutionary biology↗

bioRxiv2025

AC electro-osmosis in bacterial biofilms: a cautionary tale for electrophysiology experiments

Synthetic cationic fluorophores are used widely as probes to measure the membrane potentials of bacterial cells, eukaryotic cells and organelles, such as mitochondria. An external oscillating electric field was applied to Escherichia coli cells using microelectrodes and AC electro-osmosis was observed for the fluorophores, independent of the electrophysiology of the bacteria, giving rise to phantom action potentials. The fluorophores migrate around the microfluidic device in vortices modulating their concentration having decreases or dips in fluorescence. We show that the fluorescent dips are universally present when using cationic fluorophores, such as thioflavin-T, propidium iodide, Syto9 and Sytox Green, with or without E. coli cells in the inoculum, when stimulated with AC voltages. This is in contrast to the study of Stratford et al (PNAS, 2019) who claim the existence of action potentials. Furthermore, E. coli biofilms also demonstrated similar phenomena with dips in the fluorescence. We measured the relaxation times of the fluorophores experiencing AC electro-osmosis, which depended on the biofilm, the cells and the fluorophores used. PI had the smallest relaxation time and Syto9 the highest. Removing the cells resulted in longer relaxation times and introducing biofilm did not significantly change the relaxation times compared with the single cell experiments. Furthermore, fluorescently labelled DNA and fluorescent colloidal beads also demonstrate fluorescent dips through AC electro-osmosis, showing that these particles can be driven through biofilms. This is the first study of AC electro-osmosis in bacterial biofilms, indicating a surprisingly high mobility of charged molecules within the extracellular polymeric substance, which could be used to treat biofilms i.e. to increase the kinetics of delivery of antibiotics.

biophysics↗

bioRxiv2023

Electrical impedance spectroscopy with bacterial biofilms: neuronal-like behaviour

Negative capacitance at low frequencies for neurons was first demonstrated in 1941 (Kenneth S. Cole) using extracellular electrodes. The phenomenon subsequently was explained by Cole using the Hodgkin-Huxley model and is due to the activity of voltage-gated potassium ion channels. We show that E. coli biofilms exhibit significant stable negative capacitances at low frequencies when they experience a small DC bias voltage in electrical impedance spectroscopy experiments. Using a frequency domain Hodgkin-Huxley model, we characterize the conditions for the emergence of this feature and demonstrate that the negative capacitance exists only in biofilms containing living cells. Furthermore, we established the importance of the voltage-gated potassium ion channel, Kch, using knock-down mutants. The experiments provide further evidence for voltage-gated ion channels in E. coli and a new, low-cost method to probe biofilm electrophysiology e.g. to understand the efficacy of antibiotics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/568527v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@e3df05org.highwire.dtl.DTLVardef@46c5d2org.highwire.dtl.DTLVardef@6572a6org.highwire.dtl.DTLVardef@4a104a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Electrical Signalling in Three Dimensional Bacterial Biofilms Using an Agent Based Fire-Diffuse-Fire Model

Agent based models were used to describe electrical signalling in bacterial biofilms in three dimensions. Specifically, wavefronts of potassium ions in E. coli biofilms subjected to stress from blue light were modelled from experimental data. Electrical signalling only occurs when the biofilms grow beyond a threshold size, which we have shown to vary with the K+ ion diffusivity and the K+ ion threshold concentration which triggered firing in the fire-diffuse-fire model. The transport of the propagating wavefronts shows super-diffusive scaling on time. K+ ion diffusivity is the main factor that affects the wavefront velocity. The K+ ion diffusivity and the firing threshold also affect the anomalous exponent for the propagation of the wavefront determining whether the wavefront is sub-diffusive or super-diffusive. The geometry of the biofilm and its relation to the mean square displacement (MSD) of the wavefront as a function of time was investigated for spherical, cylindrical, cubical and mushroom-like structures. The MSD varied significantly with geometry; an additional regime to the kinetics occurred when the potassium wavefront leaves the biofilm. Adding cylindrical defects to the biofilm, which are known to occur in E. coli biofilms, the wavefront MSD also had an extra kinetic regime for the propagation through the defect.

biophysics↗

bioRxiv2023

Working together to control mutation: how collective peroxide detoxification determines microbial mutation rate plasticity

Mutagenesis is responsive to many environmental factors. Evolution therefore depends on the environment not only for selection but also in determining the variation available in a population. One such environmental dependency is the inverse relationship between mutation rates and population density in many microbial species. Here we determine the mechanism responsible for this mutation rate plasticity. Using dynamical computational modelling and in vivo mutation rate estimation we show that the negative relationship between mutation rate and population density arises from the collective ability of microbial populations to control concentrations of hydrogen peroxide. We demonstrate a loss of this density-associated mutation rate plasticity when Escherichia coli populations are deficient in the degradation of hydrogen peroxide. We further show that the reduction in mutation rate in denser populations is restored in peroxide degradation-deficient cells by the presence of wild-type cells in a mixed population. Together, these model-guided experiments provide a mechanistic explanation for density-associated mutation rate plasticity, applicable across all domains of life, and frames mutation rate as a dynamic trait shaped by microbial community composition.

evolutionary biology↗

bioRxiv2023

Environmental and genetic influence on rate and spectrum of spontaneous mutations in Escherichia coli

Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterised the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a {Delta}luxS deletant strain --a gene with roles in both quorum sensing and the recycling of methylation products used in Escherichia colis DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive {Delta}luxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.

evolutionary biology↗

bioRxiv2022

Emergence of ion-channel mediated electrical oscillations in E. coli biofilms

Bacterial biofilms are communities of bacteria usually attached to solid strata and often differentiated into complex structures. Communication across biofilms has been shown to involve chemical signaling and, more recently, electrical signaling in Gram positive biofilms. We report for the first time, community-level synchronized membrane potential dynamics in three-dimensional E. coli biofilms. Two hyperpolarization events are observed in response to light stress. The first requires mechanically sensitive ion channels (MscK, MscL and MscS) and the second needs the Kch-potassium channel. The channels mediated both local spiking of single E. coli biofilms and long-range coordinated electrical signaling in E. coli biofilms. The electrical phenomena are explained using Hodgkin-Huxley and 3D fire-diffuse-fire agent-based models. These data demonstrate that electrical wavefronts based on potassium ions are a mechanism by which signaling occurs in Gram negative biofilms and as such may represent a conserved mechanism for communication across biofilms.

biophysics↗

bioRxiv2018

Opposing effects of population density and stress on Escherichia coli mutation rate

Evolution depends on mutations. For an individual genotype, the rate at which mutations arise is known to increase with various stressors (stress-induced mutagenesis - SIM) and decrease at high population density (density-associated mutation-rate plasticity - DAMP). We hypothesised that these two forms of mutation rate plasticity would have opposing effects across a nutrient gradient. Here we test this hypothesis, culturing Escherichia coli bacteria in increasingly rich media. We distinguish an increase in mutation rate with added nutrients through SIM (dependent on error-prone polymerases Pol IV and Pol V) and an opposing effect of DAMP (dependent on MutT, which removes oxidised G nucleotides). The combination of DAMP and SIM result in a mutation rate minimum at intermediate nutrient levels (which can support 7x108 cells ml-1). These findings demonstrate a strikingly close and nuanced relationship of ecological factors - stress and population density - with mutation, the fuel of all evolution.

evolutionary biology↗