Rok Krašovec

Mutation rate plasticity in rifampicin resistance depends on Escherichia coli cell–cell interactions

Variation of mutation rate at a particular site in a particular genotype, in other words mutation rate plasticity (MRP), can be caused by stress or ageing. However, mutation rate control by other factors is less well characterized. Here we show that in wild-type Escherichia coli (K-12 and B strains), the mutation rate to rifampicin resistance is plastic and inversely related to population density: lowering density can increase mutation rates at least threefold. This MRP is genetically switchable, dependent on the quorum-sensing gene luxS—specifically its role in the activated methyl cycle—and is socially mediated via cell–cell interactions. Although we identify an inverse association of mutation rate with fitness under some circumstances, we find no functional link with stress-induced mutagenesis. Our experimental manipulation of mutation rates via the social environment raises the possibility that such manipulation occurs in nature and could be exploited medically.

On the origin of cancer: Can we ignore coherence?

A growing number of inconsistencies have accumulated within the genetically deterministic paradigm of the origin of cancer. Among them the most important are the nonspecific nature of cancer mutations and the non-cell-autonomous factors of cancer initiation and progression. Epigenetic aspects of cancer and cancer systems biology represent novel approaches to cancer aetiology and converge in the notion that cancer is characterized by a nonspecific progressive destabilization of multiple molecular pathways. The coherent behaviour of certain cellular subsystems has been theoretically predicted for a long time to have a general role in coordinating biological processes. However, it has only recently gained major scientific interest when it was measured on photosynthetic complexes at physiological temperatures and confirmed to have a direct effect over the dynamics of the energy transfer. Several theoretical and experimental considerations suggest that cancer might be associated with the absence or impairment of the proper coherent dynamics in certain biological structures, most notably in the microtubules. We review those models and suggest that impaired coherence might largely contribute to the progressive destabilization of the molecular and gene regulatory networks, thus connecting different non-genetic aspects of cancer.

Bacterial multicellularity as a possible source of antibiotic resistance

Knowledge about survival of micro-organisms in stressful situations not only influences the evolutionary theory in a fundamental way, but bears an extraordinary importance in finding a global solution to a very concrete urgent problem of mankind, namely bacterial resistance to antibiotics. Recent in vitro experiments demonstrate that the adaptive mutation process involving transient hypermutators could be one of the most important mechanisms whereby bacterial cells achieve the antibiotic resistance. An effective response of the mutation rates to specific selective conditions and an increasing number of conclusive evidence that bacterial cells are indeed communicative and co-operative organisms lead us to a hypothesis that the emergence of the antibiotic resistant mutants through the so-called adaptive mutation is deeply connected with the multicellular organisation of bacterial cells.

Possible time-dependent effect of ions and hydrophilic surfaces on the electrical conductivity of aqueous solutions.

The purpose of this work was to determine the influence of mechanical and electrical treatment on the electrical conductivity of aqueous solutions. Solutions were treated mechanically by iteration of two steps: 1:100 dilution and vigorous shaking. These two processes were repeated until extremely dilute solutions were obtained. For electrical treatment the solutions were exposed to strong electrical impulses. Effects of mechanical (as well as electrical) treatment could not be demonstrated using electrical conductivity measurements. However, significantly higher conductivity than those of the freshly prepared chemically analogous solutions was found in all aged solutions except for those samples stored frozen. The results surprisingly resemble a previously observed weak gel-like behavior in water stored in closed flasks. We suggest that ions and contact with hydrophilic glass surfaces could be the determinative conditions for the occurrence of this phenomenon.

Spontaneous mutation rate is a plastic trait associated with population density across domains of life

Rates of random, spontaneous mutation can vary plastically, dependent upon the environment. Such plasticity affects evolutionary trajectories and may be adaptive. We recently identified an inverse plastic association between mutation rate and population density at 1 locus in 1 species of bacterium. It is unknown how widespread this association is, whether it varies among organisms, and what molecular mechanisms of mutagenesis or repair are required for this mutation-rate plasticity. Here, we address all 3 questions. We identify a strong negative association between mutation rate and population density across 70 years of published literature, comprising hundreds of mutation rates estimated using phenotypic markers of mutation (fluctuation tests) from all domains of life and viruses. We test this relationship experimentally, determining that there is indeed density-associated mutation-rate plasticity (DAMP) at multiple loci in both eukaryotes and bacteria, with up to 23-fold lower mutation rates at higher population densities. We find that the degree of plasticity varies, even among closely related organisms. Nonetheless, in each domain tested, DAMP requires proteins scavenging the mutagenic oxidised nucleotide 8-oxo-dGTP. This implies that phenotypic markers give a more precise view of mutation rate than previously believed: having accounted for other known factors affecting mutation rate, controlling for population density can reduce variation in mutation-rate estimates by 93%. Widespread DAMP, which we manipulate genetically in disparate organisms, also provides a novel trait to use in the fight against the evolution of antimicrobial resistance. Such a prevalent environmental association and conserved mechanism suggest that mutation has varied plastically with population density since the early origins of life.

Monotonicity of fitness landscapes and mutation rate control

A common view in evolutionary biology is that mutation rates are minimised. However, studies in combinatorial optimisation and search have shown a clear advantage of using variable mutation rates as a control parameter to optimise the performance of evolutionary algorithms. Much biological theory in this area is based on Ronald Fisher’s work, who used Euclidean geometry to study the relation between mutation size and expected fitness of the offspring in infinite phenotypic spaces. Here we reconsider this theory based on the alternative geometry of discrete and finite spaces of DNA sequences. First, we consider the geometric case of fitness being isomorphic to distance from an optimum, and show how problems of optimal mutation rate control can be solved exactly or approximately depending on additional constraints of the problem. Then we consider the general case of fitness communicating only partial information about the distance. We define weak monotonicity of fitness landscapes and prove that this property holds in all landscapes that are continuous and open at the optimum. This theoretical result motivates our hypothesis that optimal mutation rate functions in such landscapes will increase when fitness decreases in some neighbourhood of an optimum, resembling the control functions derived in the geometric case. We test this hypothesis experimentally by analysing approximately optimal mutation rate control functions in 115 complete landscapes of binding scores between DNA sequences and transcription factors. Our findings support the hypothesis and find that the increase of mutation rate is more rapid in landscapes that are less monotonic (more rugged). We discuss the relevance of these findings to living organisms.

Deep Significance of the Field Concept in Contemporary Biomedical Sciences

Since antiquity, biology has had two opposing views of life and organisms: holistic (organismic) and reductionist. In contemporary biology, the molecular reductionist approach prevails—its central entity being the gene. Organicism lingers on the margin of biology, having well-elaborated ideas but no empirical confirmation for the integrative biological entity. The latter could be found in the endogenous coherent EM field (ECEMF), since it organizes countless cellular processes, including cells division, and through the coupling of coherence domains integrates the whole organism. A serious and thorough reconsideration of life and organisms in light of this new biological entity would have far-reaching consequences in all areas of biological science, i.e., in ontogeny, the theory of evolution, understanding and combating serious illnesses, and above all, cancer.

Where antibiotic resistance mutations meet quorum-sensing

We do not need to rehearse the grim story of the global rise of antibiotic resistant microbes. But what if it were possible to control the rate with which antibiotic resistance evolves by de novo mutation? It seems that some bacteria may already do exactly that: they modify the rate at which they mutate to antibiotic resistance dependent on their biological environment. In our recent study [Krašovec, et al. Nat. Commun. (2014), 5, 3742] we find that this modification depends on the density of the bacterial population and cell-cell interactions (rather than, for instance, the level of stress). Specifically, the wild-type strains of Escherichia coli we used will, in minimal glucose media, modify their rate of mutation to rifampicin resistance according to the density of wild-type cells. Intriguingly, the higher the density, the lower the mutation rate (Figure 1). Why this novel density-dependent ‘mutation rate plasticity’ (DD-MRP) occurs is a question at several levels. Answers are currently fragmentary, but involve the quorum-sensing gene luxS and its role in the activated methyl cycle.

Critical Mutation Rate has an Exponential Dependence on Population Size for Eukaryotic-Length Genomes

The critical mutation rate (CMR) determines the shift between survival-of-the-fittest and the survival of individuals with greater mutational robustness (the flattest). Small populations are more likely to exceed the CMR and become less well adapted; understanding the CMR is crucial to understanding the potential fate of small populations under threat of extinction. Here we present a simulation model capable of utilising input parameter values within a biologically relevant range. A previous study identified an exponential fall in CMR with decreasing population size, but the parameters and output were not directly relevant outside artificial systems. The first key contribution of this study is the identification of an inverse relationship between CMR and gene length when the gene length is comparable to that found in biological populations. The exponential relationship is maintained, and the CMR is lowered to between two to five orders of magnitude above existing estimates of per base mutation rate for a variety of organisms. The second key contribution of the study is the identification of an inverse relationship between CMR and the number of genes. Using a gene number in the range for Arabidopsis thaliana produces a CMR close to its known mutation rate; per base mutation rates for other organisms are also within one order of magnitude. This is the third key contribution of the study as it represents the first time such a simulation model has used input and produced output both within range for a given biological organism. This novel convergence of CMR model with biological reality is of particular relevance to populations undergoing a bottleneck, under stress, and subsequent conservation strategy for populations on the brink of extinction.

Conductivity measurements as a possible means to measure the degree of water ordering

In our extended experimental work with aqueous solutions of NaHCO3 we noticed higher electrical conductivity in aged solutions in comparison to the conductivity of chemically analogous fresh (one day old) solutions. The phenomenon was found in solutions left undisturbed for longer time (~ 1 year). Most probably the dissolved ions as well as the contact with hydrophilic surfaces play the essential role. We found that higher conductivity is proportional to higher surface vs. volume ratio.