Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus.

Abstract

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus. When iolQ was inactivated in G. kaustophilus, not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5'-RGWAAGCGCTTSCY-3' (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.