Various colorimetric indicators have been developed that allow one to easily dis

Question

Various colorimetric indicators have been developed that allow one to easily distinguish between gal + and gal –. These can be used, for example, to measure mutagenesis rates of a gal + specialized transducing phage that carries the gal genes. e.g., Tetrazolium in the appropriate media will cause gal + to make a red plaque on an appropriate E. coli host, compared to a colorless plaque for gal-. To measure mutagenesis rates of the E. coli host bacteria, there are many assays that can easily be applied (e.g., the rate of colonies that appear that are resistant to an antibiotic.).

An E. coli culture is lightly irradiated with UV light such that ~98% of the cells are still able to make colonies. Among the survivors, we find that the rate of mutation to antibiotic resistance has increased 50-fold relative to an un-irradiated culture. Interestingly, if we grow (un-irradiated) gal + on the irradiated and un-irradiated cells, the rate of gal – plaques is also increased 50-fold by the irradiated host even though the phage saw no damaging UV! What is the explanation of this effect?

Explanation / Answer

Based on the above results of an experiment, we can suggest the following explanation.

When E. coli was lightly irradiated with UV light, it accumulated mutations in the promoter region upstream of both antibiotic resistance gene and genes encoding for restriction enzymes in E. coli resulting in enhanced gene expression of both antibiotic resistance gene and genes encoding for restriction enzymes. An enhanced gene expression relates with an increase in transcription and translation and therefore, resulting in larger amounts of protein.

The restriction enzymes produced by E.coli are known to digest the un-methylated DNA. When gal + attacks the E.coli, DNA of gal + phage (un-methylated and contain gal gene) is digested by restriction enzymes which prevents the synthesis of gal protein. The absence of gal protein will prevent the hydrolysis of tetrazolium to red colour. Therefore, restriction digestion of gal + DNA will result in the white/ gal- plaques in irradiated E.coli.

The mutations that resulted in 50-fold increase in antibiotic resistance will thus increase the production of restriction enzymes and hence will account for 50-fold increase in gal- plaques in irradiated host.

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