General stress proteins: Novel function and signals for induction of stationary phase genes in E.coli
Survival during conditions when nutrients become scarce requires adaptation and expression of genes for maintenance in order for the cell to survive. Among the numerous proteins involved in adaptation and regulation under these conditions, the stationary phase sigma factor, σS, and the Universal stress proteins contribute to survival and bestow the cell with general stress protective functions during growth arrest. In this work we found new mechanisms for the cell to prepare and sense the intracellular environment and respond accordingly. The usp genes and the rpoS gene (encoding σS) were found to be positively regulated by metabolic intermediates of the glycolysis in the central metabolic pathway. Specifically, mutations and conditions resulting in fructose-6-phosphate accumulation elicit superinduction of these genes upon carbon starvation, whereas genetic manipulations reducing the pool size of fructose-6-phosphate have the opposite effect. Under carbon starvation, transcription of the usp and the rpoS genes require and are modulated by the alarmone ppGpp. The observed positive transcriptional regulation by fructose-6-phosphate is not via alterations of the levels of ppGpp. None of the known regulators examined were found to be required for the superinduction. We suggest a novel regulatory mechanism involving the phosphorylated intermediates as a signal molecule for monitoring and subsequent regulation of stress defense genes. Based on mutational studies we also suggest that this signaling mechanism secures accumulation of required survival proteins preceding the complete depletion of the external carbon source. Entry into stationary phase promotes a dramatic stabilization on the sigma factor σS. Mistranslated and oxidized proteins were shown to contribute to elevated levels of σS and transcription of its regulon. Furthermore, ribosomal alleles with enhanced translational accuracy attenuate induction of the RpoS regulon and prevent stabilization of σS. Destabilization of σS is governed by the ClpXP protease, for which aberrant proteins also are substrates. Mechanistically, generation of mistranslated proteins by starvation, or other means, competes for the common enzyme for degradation, and thereby sequesters the pool in favor of σS stabilization. A growing body of evidence shows that there is an intimate connection between proteins required for genome stability and stationary phase survival. We show that the integral membrane protein UspB, a member of the RpoS regulon, is required for proper DNA repair as mutants lacking uspB are sensitive to several DNA damaging conditions. Genetic and biochemical studies demonstrate that UspB acts in the RuvABC recombination repair pathway and removing uspB creates a phenocopy of the DNA resolvase mutant, ruvC, which includes a reduced efficiency in resolving Holliday junctions. Further, we show that the uspB mutant phenotype can be suppressed by ectopic overproduction of RuvC and that both ruvC and uspB mutants can be suppressed by inactivating recD. The fact that RuvABC-dependent repair requires UspB for proper activity suggests that the σS-regulon works together with DNA repair pathways under stress conditions to defend the cell against genotoxic stress.
Parts of work
I; Metabolic control of the Escherichia coli universal stress protein response through fructose-6-phosphate. Persson Ö, Valadi A, Nyström T, Farewell A. Mol Microbiol. 2007 65(4):968-78.::DOI::10.1111/j.1365-2958.2007.05838.xII; The levels of fructose-6-phosphate regulate σS-dependent transcription upon entry to stationary phase in E. coli. Persson, Ö, Gummesson, B., Hallberg, E., Lilja, E., and Farewell, A. ManuscriptIII; Decline in ribosomal fidelity contributes to the accumulation and stabilization of the master stress response regulator σS upon carbon starvation. Fredriksson Å, Ballesteros M, Peterson CN, Persson Ö, Silhavy TJ, Nyström T. Genes Dev. 2007 21(7):862-74.::DOI::10.1101/gad.409407IV; UspB, a member of the sigma-S regulon, is required for RuvC-dependent resolution of Holliday junctions. Persson, Ö, Nyström, T and Farewell, A. Under revision DNA Repair.
Doctor of Philosophy
Göteborgs universitet. Naturvetenskapliga fakulteten
Department of Cell and Molecular Biology ; Institutionen för cell- och molekylärbiologi
Tidagen den 25 Maj 2010, kl. 10.00, Hörsal Arvid Carlsson, Academicum, Medicinaregatan 3
Date of defence