Chemical and UV Mutagenesis

Abstract

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast <em>Schizosaccharomyces pombe</em>

“…The protocol described here represents a novel and simple screen for spontaneous suppressor mutations detectable through phenotypic recovery of mutations conferring slow growth in fission yeast, a phenotype characteristic of over 400 genes in S. pombe, the function of many of which remains unknown 2,32 . Previous methods have taken other approaches to screen for suppressor mutations in microorganisms, including the use of mutagens 21 , or the application of a temperature shift in temperature-sensitive mutant backgrounds 33 . In contrast, this protocol shows that phenotypic recovery occurs without additional environmental/chemical interference and highlights the fitness advantage of the rise of suppression mutations eventually taking over the resources available in liquid culture.…”

“…In addition, manganese chloride has long been used in yeasts for the ability of the manganese cation to inhibit DNA repair pathways 20 . Another common approach is UV-induced mutagenesis, which generates genome-wide mutagenic pyrimidine dimers 21,22 .…”

Oxygen activation by mononuclear nonheme iron dioxygenases involved in the degradation of aromatics

“…Time-resolved advanced spectroscopy and X-ray crystallography, site-directed alteration ( i.e . substitution of a natural or unnatural amino acid) or the novel chemical mutagenesis approaches [104–106], and synthetic techniques for mechanism-based probes are expected to yield fundamental structural and chemical insights into O 2 activation and insertion for mechanistic enzymology of the non-heme Fe dioxygenases.…”

Enhancement of clavulanic acid production by mutant Streptomyces clavuligerus

“…Chemical mutagenesis can be performed using a variety of chemicals that cause DNA damage, leading to single nucleotide changes and/or deletions, while UV light leads to DNA damage both directly and through the generation of reactive oxygen species. 5 Most Streptomyces species studied are unstable for certain characters (i.e., a spontaneous mutation rate higher than 0.1% per spore). The chromosome is very unstable and undergoes very large deletions spontaneously.…”

“…UV is a very convenient and relatively safe method, and in the range of 200-300nm it produces thymidine dimmers and increases the probability of deletion during the duplication process. 8 UV irradiation can also increase the concentration of CA up to two-fold, 5,9 as it also makes their production cheaper. These molecular changes affect different phenotypical properties, often pleiotropically, including morphological differentiation, production of secondary metabolites (pigments and antibiotics), antibiotic resistance, secretion of extracellular enzymes and sometimes genes for primary metabolism, particularly one or more steps in the arginine biosynthetic pathway.…”