Clp dependent proteolysis and the role of the McsB arginine kinase in the protein quality control network of low GC Gram+ bacteria

• Applicant: Dr. Ulf Gerth
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 278600295

Proteins are usually born at the ribosome and after an individual lifespan, which can vary between minutes, hours or even days they are destroyed by proteolytic machines in most cases due to functional deficiency. Clp proteases represent the most important intracellular proteolytic systems with the protease ClpP as the major determinant of bulk protein turnover in Bacillus subtilis. The ATP-dependent ClpCP protease is mainly responsible for the degradation of heat-damaged proteins, the heat-sensitive transcriptional repressor CtsR as well as biosynthetic enzymes during glucose starvation in B. subtilis. ClpXP appears to be important for the rescue of stalled ribosomes under standard growth conditions by proteolysis of ssrA-tagged proteins as well as for "secondarily-induced" oxidative stress regulators like B. subtilis MgsR and Spx. Recently, we and others described a protein arginine phosphorylation exerted by the arginine kinase McsB in B. subtilis, which acts as an adaptor protein for the ClpCP protease. Arginine phosphorylation seems to play a crucial and so far underestimated role in Gram+ bacteria. The revelation of a link between arginine phosphorylation, lifespan of proteins and protease activity is a new and compelling strategy within the ATP-dependent proteolysis research in low GC Gram+ bacteria. This grant proposal explores the: 1.) McsB-dependent mechanism of in vivo recognition and degradation of the protein quality control regulator CtsR, 2.) role of McsB-mediated arginine phosphorylation during different stress and starvation conditions using specific trap mutants, 3.) putative existence of phosphoarginine proteins in mcsB lacking bacteria such as Lacto- or Streptococci as well as complementation studies with the L. lactis ClpC ATPase will shed some light on a strict requirement of McsB for a ClpCL.lactis-dependent degradation of CtsR in B. subtilis, 4.) Clp-dependent degradation of selected "useless" and "unemployed" proteins such as the intrinsically inactive glutamate dehydrogenase GudB and gluconeogenetic GapB under non-gluconeogenetic growth conditions, 5.) connections between CtsR and the generell stress regulator SigmaB (degradation profile of the RsbW-SigmaB complex, detailed analyses to the fate of the MgsR and Spx oxidative regulators during stress and the influence of McsB arginine kinase, a putative relation between the MazEF toxin-antitoxin system, SigmaB and Clp proteolysis).

• DFG Programme: Research Grants