Activation of silent gal genes in the lac-gal regulon of Streptococcus thermophilus

Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free s ugar, Nevertheless, sequence analysis and complementation studies with Esch erichia call demonstrated that strain CNRZ 302 contained structurally intac t genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regul ator gene, galR, and followed by a galM gene and the lactose operon lacSZ, Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ30 2G, the galKTE genes were well expressed in cells grown on lactose or galac tose. In both CNRZ 302 and the Gal(+) mutant NZ302G, the transcription of t he galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis o f the gal promoter regions of NZ302G and nine other independently isolated Gal(+) mutants of CNRZ 302 revealed mutations at three positions in the gal K promoter region, which included substitutions at positions -9 and -15 as well as a single-base pair insertion at position -37 with respect to the ma in transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated pr omoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.