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 dehydrogenase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (gudBCR) in the laboratory strain 168 due to a perfect 18 bp-long direct repeat that renders the GudB enzym...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2015-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00758/full |
| _version_ | 1818590036155695104 |
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| author | Lorena eStannek Katrin eGunka Rachel eCare Ulf eGerth Fabian Moritz Commichau |
| author_facet | Lorena eStannek Katrin eGunka Rachel eCare Ulf eGerth Fabian Moritz Commichau |
| author_sort | Lorena eStannek |
| collection | DOAJ |
| description | The Gram-positive model bacterium Bacillus subtilis contains two glutamate dehydrogenase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (gudBCR) 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 GudBCR protein can hardly be detected in B. subtilis as it is rapidly degraded within stationary growth phase. Its high instability qualifies GudBCR as a model substrate for studying protein turnover in B. subtilis. Recently, we have developed a visual screen to monitor the GudBCR stability in the cell using a GFP-GudBCR fusion. Using fluorescent microscopy we found that the GFP protein is simultaneously degraded together with GudBCR. This allows us to analyze the stability of GudBCR 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-GudBCR fusion. We observed, that disruption of the arginine kinase encoding gene mcsB upon transposon insertion leads to increased amounts of the GFP-GudBCR fusion in this mutant. Deletion of the cognate arginine phosphatase YwlE in contrast results in reduced levels of the GFP-GudBCR fusion. Recently, it was shown that the kinase McsB is involved in phosphorylation of GudBCR on arginine residues. Here we show that selected arginine-lysine point mutations of GudBCR 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 GudBCR protein in B. subtilis. |
| first_indexed | 2024-12-16T09:50:09Z |
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| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-16T09:50:09Z |
| publishDate | 2015-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-ee3e6f6821354b97b8289e93dddd07b52022-12-21T22:36:06ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2015-01-01510.3389/fmicb.2014.00758127667Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilisLorena eStannek0Katrin eGunka1Rachel eCare2Ulf eGerth3Fabian Moritz Commichau4University of GöttingenUniversity of GöttingenUniversity of GöttingenUniversity of GreifswaldUniversity of GöttingenThe Gram-positive model bacterium Bacillus subtilis contains two glutamate dehydrogenase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (gudBCR) 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 GudBCR protein can hardly be detected in B. subtilis as it is rapidly degraded within stationary growth phase. Its high instability qualifies GudBCR as a model substrate for studying protein turnover in B. subtilis. Recently, we have developed a visual screen to monitor the GudBCR stability in the cell using a GFP-GudBCR fusion. Using fluorescent microscopy we found that the GFP protein is simultaneously degraded together with GudBCR. This allows us to analyze the stability of GudBCR 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-GudBCR fusion. We observed, that disruption of the arginine kinase encoding gene mcsB upon transposon insertion leads to increased amounts of the GFP-GudBCR fusion in this mutant. Deletion of the cognate arginine phosphatase YwlE in contrast results in reduced levels of the GFP-GudBCR fusion. Recently, it was shown that the kinase McsB is involved in phosphorylation of GudBCR on arginine residues. Here we show that selected arginine-lysine point mutations of GudBCR 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 GudBCR protein in B. subtilis.http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00758/fullGlutamate DehydrogenaseProtein FoldingProteolysisProtein modificationarginine phosphorylation |
| spellingShingle | Lorena eStannek Katrin eGunka Rachel eCare Ulf eGerth Fabian Moritz Commichau Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis Frontiers in Microbiology Glutamate Dehydrogenase Protein Folding Proteolysis Protein modification arginine phosphorylation |
| title | Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis |
| title_full | Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis |
| title_fullStr | Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis |
| title_full_unstemmed | Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis |
| title_short | Factors that mediate and prevent degradation of the inactive and unstable GudB protein in Bacillus subtilis |
| title_sort | factors that mediate and prevent degradation of the inactive and unstable gudb protein in bacillus subtilis |
| topic | Glutamate Dehydrogenase Protein Folding Proteolysis Protein modification arginine phosphorylation |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00758/full |
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