Development of a device that generates a temperature gradient in a microtiter plate for microbial culture.

Although temperature is a fundamental parameter in biology, testing various temperature conditions simultaneously is often difficult. In the present study, we developed a device for generating a temperature gradient in arrays of wells on a microtiter plate. This device consists of a pair of Peltier elements and temperature sensors placed on both ends of a flat aluminum bar to generate a linear temperature gradient. The device loads a microtiter plate at the center of the aluminum bar and transfers the temperature gradient to the bottom of the wells in the plate. This device successfully maintained a temperature gradient of 38.2 to 43.1°C on the horizontal axis of a 96-well microtiter plate in an incubator at 31°C. Furthermore, using this device, we demonstrated a laboratory evolution experiment of Escherichia coli, which was selected on the basis of its ability to grow at high temperatures. The developed device also facilitates a two-dimensional assay to determine the effects of temperature and drug concentrations on cellular growth.

Mutational property of newly identified mutagen l-glutamic acid γ-hydrazide in Escherichia coli.

We previously found that an l-glutamine analog l-glutamic acid γ-hydrazide has high mutagenic activity through the high-throughput laboratory evolution of Escherichia coli. In this study, mutagenicity and mutational property of l-glutamic acid γ-hydrazide were examined by the Ames test and mutation accumulation experiments using E. coli. The Ames test revealed that l-glutamic acid γ-hydrazide showed higher mutagenic activity without metabolic activation than known mutagens 2-aminoanthracene, and cobalt(II) acetate tetrahydrate. This result indicates that l-glutamic acid γ-hydrazide does not require metabolic activation for mutagenic activity in E. coli. Mutation accumulation experiments and whole-genome sequencing analysis revealed the number and spectrum of the accumulated mutations with or without l-glutamic acid γ-hydrazide. In the presence of l-glutamic acid γ-hydrazide, MDS42 strain accumulated 392.3 ± 116.2 point mutations during 30 passages corresponding to 777 generations, while MDS42 strain accumulated 1.5 ± 2.5 point mutations without l-glutamic acid γ-hydrazide during 50 passages corresponding to 1341 generations. The mutational spectrum of l-glutamic acid γ-hydrazide was G/C to A/T transition (82.2 ± 4.3 %) and A/T to G/C transition (17.4 ± 4.3 %). These results indicated that l-glutamic acid γ-hydrazide has a strong mutagenic activity.