Effect of Non-Lethal Selection on Spontaneous Revertants of Frameshift Mutations: The Escherichia coli hisF Case

Abstract

Microorganisms possess the potential to adapt to fluctuations in environmental parameters, and their evolution is driven by the continuous generation of mutations. The reversion of auxotrophic mutations has been widely studied; however, little is known about the reversion of frameshift mutations resulting in amino acid auxotrophy and on the structure and functioning of the protein encoded by the revertant mutated gene. The aims of this work were to analyze the appearance of reverse mutations over time and under different selective pressures and to investigate revertant enzymes’ three-dimensional structures and their correlation with a different growth ability. Escherichia coli FB182 strain, carrying the hisF892 single nucleotide deletion resulting in histidine auxotrophy, was subjected to different selective pressures, and revertant mutants were isolated and characterized. The obtained results allowed us to identify different indels of different lengths located in different positions in the hisF gene, and relations with the incubation time and the selective pressure applied were observed. Moreover, the structure of the different mutant proteins was consistent with the respective revertant ability to grow in absence of histidine, highlighting a correlation between the mutations and the catalytic activity of the mutated HisF enzyme.

Genomic Analysis of Endophytic Bacillus-Related Strains Isolated from the Medicinal Plant Origanum vulgare L. Revealed the Presence of Metabolic Pathways Involved in the Biosynthesis of Bioactive Compounds

“…Each curve was performed in triplicate. The calculation of the areas under the growth curve (AUC) was obtained according to the chained trapezoidal rule [ 14 ]. Statistical significance of the differences between each condition was calculated through a non-parametric test and a post hoc analysis, Kruskal-Wallis and Dunn tests.…”

Application of Cloning-Free Genome Engineering to Escherichia coli

“…In order to check this hypothesis, the nucleotide sequence of each amplicon was Sanger sequenced, and the data obtained are summarized as follows: Seven revertants owned a wild-type hisF gene replacing the E. coli FB182 mutated one (group A). Based on the previous assumption (i.e., the very low probability, less than 4%, of spontaneous restoring of the correct sequence) [ 21 ], they should be the result of a recombination event involving a single copy of the wild-type hisF gene; Thirty-two revertants possessed two or more in tandem hisF copies (group B) and are the result of recombinational events involving one, two, or more copies of the donor DNA; Just one transformant was a chromosomal revertant owning a hisF gene with a restored frame but a different sequence from the wild-type one (group C), according to Del Duca et al [ 21 ]. …”

“…Seven revertants owned a wild-type hisF gene replacing the E. coli FB182 mutated one (group A). Based on the previous assumption (i.e., the very low probability, less than 4%, of spontaneous restoring of the correct sequence) [ 21 ], they should be the result of a recombination event involving a single copy of the wild-type hisF gene;…”

“…The E. coli mutant strain FB182 ( hisF892 ), transformed with the circularized E. coli hisF gene, was chosen as a model for the present study. This choice relies on recent data from Del Duca et al [ 21 ], who analyzed the frequency and type of E. coli FB182 natural reverse mutations under selective pressure conditions (i.e., absence of histidine in the culture medium). Data obtained revealed that only a very low percentage of the HisF + revertants restored the wild-type genotype (3.8%).…”

Microbial Genetics and Evolution

“…They demonstrated that somatic assortment in P. tetraurelia occurs rather slowly, being prevented by the high ploidy of its macronucleus. In the second work, a histidine-auxotrophic mutant strain of E. coli , having a single-nucleotide deletion in its hisF gene, was used to study the effects of selective pressure on the reversion of frameshift mutations through a directed-evolution experiment [ 18 ]. The frequency of revertant mutants and the kind of mutation were finally correlated to the intensity of selective pressure, cultivation time, the tertiary structure of HisF, and ability to grow in the absence of His.…”