Factors That Mediate and Prevent Degradation of the Inactive and Unstable GudB Protein in Bacillus Subtilis

The Gram-positive model bacterium Bacillus subtilis contains two glutamate dehydro genase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (gudB(CR) ) in the laboratory strain 168 due to a perfect 18 bp-long direct repeat that renders the GudB enzyme inactive and unstable. Although constitutively expressed the GudB(CR) protein can hardly be detected in B. subtilis as it is rapidly degraded within stationary growth phase. Its high instability qualifies GudB(CR) as a model substrate for studying protein turnover in B. subtilis. Recently, we have developed a visual screen to monitor the GudB(CR) stability in the cell using a GFP-GudB(CR) fusion. Using fluorescent microscopy we found that the GFP protein is simultaneously degraded together with GudB(CR). This allows us to analyze the stability of GudB(CR) in living cells. By combining the visual screen with a transposon mutagenesis approach we looked for mutants that show an increased fluorescence signal compared to the wild type indicating a stabilized GFP-GudB(CR) fusion. We observed, that disruption of the arginine kinase encoding gene mcsB upon transposon insertion leads to increased amounts of the GFP-GudB(CR) fusion in this mutant. Deletion of the cognate arginine phosphatase YwlE in contrast results in reduced levels of the GFP-GudB(CR) fusion. Recently, it was shown that the kinase McsB is involved in phosphorylation of GudB(CR) on arginine residues. Here we show that selected arginine-lysine point mutations of GudB(CR) exhibit no influence on degradation. The activity of McsB and YwlE, however, are crucial for the activation and inhibition, respectively, of a proteolytic machinery that efficiently degrades the unstable GudB(CR) protein in B. subtilis.