Using the gel mobility-shift assay with protein fractions from different developmental stages of solid-grown Streptomyces aureofaciens, we identified two different proteins specifically bound to the whiH promoter region. Only one protein (RwhA) was detected in young substrate mycelium cultivated in liquid medium. On comparing the mobility of the resulting complexes, one of the bound proteins present in substrate mycelium and in early stages of aerial mycelium seemed to be identical with the RwhA. The other detected protein with a higher mobility (RwhB) was present in all developmental stages except for mature spores. DNA footprinting analysis localized the binding site of RwhB to nucleotides -23 to +40 relative to the transcription start point of the PwhiH promoter. RwhA from young substrate mycelium protected the DNA fragment from -106 to -77 in coding strand and -126 to -82 in noncoding strand. WhiH has homology to a large family of metabolism-related repressors and seems to regulate negatively its own expression. These observations (and the results of transcription analysis of the whiH gene obtained earlier) suggest that two different proteins influence the expression of whiH gene in S. aureofaciens. The putative repressor-like RwhA protein protects expression of whiH in substrate mycelium either in liquid medium or during differentiation. The other detected protein, RwhB, which binds to the whiH promoter region during differentiation, may represent two forms of WhiH, one with a repressor role at the beginning of differentiation and second with the role of activator at the time of sporulation.

Institute of Molecular Biology Slovak Academy of Sciences 842 51 Bratislava Slovakia

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