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J Biol Chem, Vol. 273, Issue 27, 16860-16864, July 3, 1998

Conversion of Temperature-sensitive to -resistant Gene Expression Due to Mutations in the Promoter Region of the Melibiose Operon in Escherichia coli

Eiji Tamai, Tadashi Shimamoto^§, Masaaki Tsuda, Tohru Mizushima, and Tomofusa Tsuchiya^§

From the  Department of Microbiology, Faculty of Pharmaceutical Sciences, and ^§ Gene Research Center, Okayama University, Tsushima, Okayama, 700-8530, Japan

The melibiose utilization system of Escherichia coli W3133, a derivative of K12, is nonfunctional between 37 and 42 °C. The reason for this temperature sensitivity was thought to be that the melibiose transporter (MelB) of W3133 cells was temperature-sensitive. A mutant W3133-2 has been isolated as a temperature-resistant strain that can utilize melibiose between 37 and 42 °C. However, we found that the melibiose transporter of the W3133-2 was still temperature-sensitive. Half-life activities of the melibiose transporter at 37 °C (or 40 °C) in both E. coli W3133 and W3133-2 were exactly the same. Furthermore, we found that the nucleotide sequence of coding region of the melB structural gene (the second gene of the melibiose operon) of W3133-2 was exactly the same as that of W3133. Activity of -galactosidase (product of the first gene, melA, of the melibiose operon) of W3133 cells grown at 40 °C was very low, although that of W3133-2 cells grown at 40 °C was high. These observations suggested that expression of the melibiose operon in W3133 is also temperature-sensitive. In fact, we found that the expression in W3133 cells was temperature-sensitive, while that in W3133-2 cells was temperature-resistant, by analyzing mRNA levels using the Northern blot method. Furthermore, we identified mutations in the promoter region of the melibiose operon of W3133-2 that resulted in the elongation of an 18 nucleotide inverted repeat sequence to a 28-nucleotide repeat sequence present immediately upstream of the 35 region. This may stabilize a possible stem structure due to the inverted repeat at 37-42 °C.

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