| Research Abstract |
Alanine-scanning mutagenesis of the putative HTH region of DegU was carried out. As a result, we obtained five mutants, N183A, I192A, T196A, H200A and I205A, showing expression levels similar to that in a degU-deficient mutant with respect to comG, one of the targets of ComK. Western analysis revealed the stability of the mutant DegU proteins similar to that of wild-type DegU. Examination of comK-promoter-binding of the mutant DegU proteins with gel retardation assay demonstrated that all the five mutant DegU proteins lost a DNA-binding activity. Next, we tested the effects of these mutant DegU proteins on aprE-lacZ expression. It was observed that the mutant DegU proteins tended to cause more severe reduction of aprE-lacZ expression than comG-lacZ expression. Thus, into these strains we introduced multicopy degR, which enhances stability of phosphorylated DegU, leading to an increase in aprE-lacZ expression. In this background still ten strains showed very low levels of aprE-lacZ expr
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ession. Western analysis showed that these mutant DegU proteins were stable in cells growing in sporulation medium with the exception of I204A. We found that DegU formed ladder-like and multimer complexes in gel retardation assay using the aprE promoter. The five mutant DegU proteins lacking the binding ability to the comK promoter also showed a reduced binding-affinity to the aprE promoter compared to wild-type DegU. In addition, four mutant DegU proteins, K195A, N199A, V201A and S202A showed reduced binding-affinity to the aprE promoter. We determined sequences recognized by DegU on the comK promoter by comK-lacZ fusions carrying various nucleotide changes. As a result, DegU-recognized sequence was determined to be 5'-GNNATTTA-N8-TAAATNNC-3'. The amino acids important for DNA-binding did not largely change upon phosphorylation by our alanine-scanning analysis of DegU, therefore, the DegU-recognized cis-sequences would not be changed upon phosphorylation. In the aprE promoter several candidates for the DegU-binding sequence were found and alteration of those sequences reduced DegU-dependency of the aprE-lacZ fusions. Less
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