Direct Selection for Mutators in Escherichia coli

Abstract

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10−5 in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.

Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection

“…Significant work has been done to study how the balance between adaptive and deleterious mutations in hypermutating strains determines evolutionary outcomes (1, 5-7, 12, 32, 76, 77). Both theoretical and experimental investigations suggest that hypermutators of various strengths will fix by hitchhiking with the adaptive mutations they facilitate under certain conditions (6,14,21,22). This effect has been demonstrated both in vitro and in vivo and is also consistent with the observations of the cooccurrence of hypermutators and drug resistance mutations in chronic infections in humans (23-26, 30, 35-37).…”

“…As a result, optimal mutation rates can differ between these high and low extremes and may be dynamically modified by selection, particularly when increases in mutational rate may be beneficial for adapting bacterial populations (6,(14)(15)(16)(17)(18). Supporting this hypothesis, moderately increased mutational rates are shown to be advantageous in both in vitro and in vivo competition experiments under conditions of selective bottlenecks in which sharp reductions in population numbers occur (6,7,(19)(20)(21)(22).…”

Evolutionary Mechanisms Shaping the Maintenance of Antibiotic Resistance

“…Furthermore, bacteria can acquire mutations that increase their genome-wide U typically 10 to 1000-fold -known as "mutators". In fact, it has been long known that recurrent pressure of antibiotics selects for mutator clones due to their increased ability to produce the rare mutations that can rescue bacterial populations from such high selective pressures [20]. Mutators are also known to exhibit increased ability for recombination [21].…”

Evolving concepts of tumor heterogeneity

“…This concept was initially described in Nowell’s paper where he attributes the high number of mutations in cancer genomes to waves of clonal selection [ 5 , 23 ]. Studies in bacteria and yeast imply mutator mutations confer a selective growth advantage on cells harboring these acquired mutations [ 24 , 25 ]. The current mutator hypothesis speculates that a small number of ‘driver’ alterations exist and, once acquired by somatic mutation, confer the cancer phenotype; however, seemingly insignificant ‘passenger’ mutations result via mechanisms yet to be elucidated [ 26 ].…”