Otto G. Berg

Selection of Resistant Bacteria at Very Low Antibiotic Concentrations

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.

The lac Repressor Displays Facilitated Diffusion in Living Cells

Slide and Find Transcription factors rapidly find their specific binding sites on chromosomal DNA. It has been proposed that searching is facilitated by complementing three-dimensional diffusion with one-dimensional diffusion along DNA. Such sliding on DNA has been observed in vitro, but whether and how far transcription factors slide along chromosomes in vivo is unclear. Hammar et al. (p. 1595) used single-molecule imaging to demonstrate that the lac repressor slides into its chromosomal operators in living cells. The average sliding distance was about 45 base pairs, and the repressor frequently slid over its operator before binding. The lac repressor slides along DNA in living cells, frequently passing its operator before binding. Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. Here we show that the lac repressor slides 45 ± 10 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (>90%) slides over its natural lacO1 operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 → AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty‐one independent lineages of the low‐fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be ≥ 10−7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin‐sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single‐nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.

A model for the statistical fluctuations of protein numbers in a microbial population

A model is presented for the distribution of protein molecules between the cells in a microbial population during steady-state growth. A general expression is derived under the sole assumption that each protein molecule has equal probability of joining either daughter cell at cell division, i.e. a binomial partitioning. With reasonable assumptions about transcription and translation probabilities a specific protein distribution is described. The main result is that this distribution is very broad, especially for small protein numbers; it is definitely not a Poisson distribution. The case of repressor proteins, which have one strong binding site on the chromosome, is treated separately. As a specific example, the distribution of β-galactosidase is described, also in regard to the influence from the statistical fluctuations in the distribution of the lac repressors.

Genomic buffering mitigates the effects of deleterious mutations in bacteria

The relationship between the number of randomly accumulated mutations in a genome and fitness is a key parameter in evolutionary biology. Mutations may interact such that their combined effect on fitness is additive (no epistasis), reinforced (synergistic epistasis) or mitigated (antagonistic epistasis). We measured the decrease in fitness caused by increasing mutation number in the bacterium Salmonella typhimurium using a regulated, error-prone DNA polymerase (polymerase IV, DinB). As mutations accumulated, fitness costs increased at a diminishing rate. This suggests that random mutations interact such that their combined effect on fitness is mitigated and that the genome is buffered against the fitness reduction caused by accumulated mutations. Levels of the heat shock chaperones DnaK and GroEL increased in lineages that had accumulated many mutations, and experimental overproduction of GroEL further increased the fitness of lineages containing deleterious mutations. These findings suggest that overexpression of chaperones contributes to antagonistic epistasis.

Selection-Driven Gene Loss in Bacteria

Gene loss by deletion is a common evolutionary process in bacteria, as exemplified by bacteria with small genomes that have evolved from bacteria with larger genomes by reductive processes. The driving force(s) for genome reduction remains unclear, and here we examined the hypothesis that gene loss is selected because carriage of superfluous genes confers a fitness cost to the bacterium. In the bacterium Salmonella enterica, we measured deletion rates at 11 chromosomal positions and the fitness effects of several spontaneous deletions. Deletion rates varied over 200-fold between different regions with the replication terminus region showing the highest rates. Approximately 25% of the examined deletions caused an increase in fitness under one or several growth conditions, and after serial passage of wild-type bacteria in rich medium for 1,000 generations we observed fixation of deletions that substantially increased bacterial fitness when reconstructed in a non-evolved bacterium. These results suggest that selection could be a significant driver of gene loss and reductive genome evolution.

Association kinetics with coupled diffusional flows: Special application to the lac repressor-operator system

The time development of the association of the lac repressor to the operator is considered in a model where the repressor is allowed to bind unspecifically to DNA and move along the DNA chain in a one-dimensional diffusion. The coupling to the three-dimensional diffusion outside the chain is introduced by letting the repressor associate and dissociate from the chain until it is finally bound to the operator. All distance correlations along the chain are included. The mean time of association is calculated and through a comparison with experimental data the molecular parameters are determined. The one-dimensional diffusion constant is found to be of the order of 10(-9) cm(2)s(-1). The model is sufficiently general to be applicable to other similar systems.

On diffusion-controlled dissociation

Abstract The theory of diffusion-controlled association is used to determine the time development of the corresponding dissociation process. The departure from a pure exponential decay will influence the entire time course, and not just the initial transient as in the case of an association process. It is also shown how this departure enters the results from temperature- jump experiments and alike. The effect is predicted to be most apparent in the case of ligand binding to a macromolecule with a large number of binding sites.

Selection of DNA binding sites by regulatory proteins.

Abstract As a consequence of progress in several fields, we are developing an understainding of the structural, thermodynamic and statistical rules that govern the binding of regulatory proteins to functional sites on the DNA genome. The current state of our understanding of these rules, and some approaches to the evolutionary selection pressures that underlie them, are summarized here.

Fluctuations and Quality of Control in Biological Cells: Zero-Order Ultrasensitivity Reinvestigated

Living cells differ from most other chemical systems in that they involve regulation pathways that depend very nonlinearly on chemical species that are present in low copy numbers per cell. This leads to a variety of intracellular kinetic phenomena that elude macroscopic modeling, which implicitly assumes that cells are infinitely large and fluctuations negligible. It is of particular importance to assess how fluctuations affect regulation in cases where precision and reliability are required. Here, taking finite cell size and stochastic aspects into account, we reinvestigate theoretically the mechanism of zero-order ultrasensitivity for covalent modification of target enzymes ( Proc. Natl. Acad. Sci. USA. 78:6840-6844). Macroscopically, this mechanism can produce a very sharp transition in target concentrations for very small changes in the activity of the converter enzymes. This study shows that the transition is much more gradual in a finite cell or a population of finite cells. It also demonstrates that the switch is exactly analogous to a thermodynamic phase transition and that ultrasensitivity is inevitably coupled to random ultravariation. As a consequence, the average response in a large population of cells will often be much more gradual than predicted from macroscopic descriptions.