Conversion of temperature-sensitive to -resistant gene expression due to mutations in the promoter region of the melibiose operon in Escherichia coli.
The melibiose utilization system of Escherichia coli W3133, a derivative of K12, is nonfunctional between 37 and 42 degreesC. 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 degreesC. However, we found that the melibiose transporter of the W3133-2 was still temperature-sensitive. Half-life activities of the melibiose transporter at 37 degreesC (or 40 degreesC) 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 alpha-galactosidase (product of the first gene, melA, of the melibiose operon) of W3133 cells grown at 40 degreesC was very low, although that of W3133-2 cells grown at 40 degreesC 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 degreesC.
Tamai
E
E
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama, 700-8530, Japan.
Shimamoto
T
T
Tsuda
M
M
Mizushima
T
T
Tsuchiya
T
T
D001483:Base Sequence; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006207:Half-Life; D008553:Melibiose / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D013696:Temperature
D002352:Carrier Proteins; D004269:DNA, Bacterial; D008553:Melibiose