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Atos-Atoc two-component signal transduction system in microorganisms - regulation of transcription of the atoDAEB operon

By Αστέριος Γρηγορούδης

Abstract

The aim of this work has been the experimental verification of the properties of the E. coli atoC gene product as the response regulator of the AtoS-AtoC two component system and transcriptional regulator of the atoDAEB operon, the products of which are responsible for the catabolism of short-chain fatty acids. These properties, as expected from the homology of the AtoC sequence with response regulators which act as part of the σ54-RNA polymerase holoenzyme, are the regulator’s phosphorylation by the sensor kinase (AtoS here), its binding to specific sequences-enhancers of the promoter, oligomerization and ATP hydrolysis, necessary for the energy coupling of the isomerization of the closed σ54-RNAP-promoter complex to an open one and subsequently the start of transcription. At first, the presence of AtoC in membrane fractions of strains where AtoS is co-expressed was verified. ΔatoSC strains’ membranes were proved negative in AtoC presence, while AtoC was only present in membrane fractions containing both components (BW28878/pUC-Az), which indicates that possibly physical contact takes place between the two proteins. In spite of the not so apparent presence of phosphorylated AtoS, the AtoC+ and AtoC- membrane fractions were both able to phosphorylate exhogenously added purified His10-AtoC. AtoC was also phosphorylated at a minimal rate when ΔatoSC E. coli BW28878 membranes were used as kinase source, which indicates a limited presence of ?cross-talk? between noncognate kinases and AtoC. As putative phosphorylation sites were identified the conserved Asp55, as a typical phosphorylaiton target and His73 which lies inside the –uncommon between response regulators- ?Η-box?. Both aminoacids were replaced by a glycine and leukine respectively, while the double mutant was also generated. Both His10- AtoCD55G and His10-AtoCH73L retained the property of being phosphorylated in vitro, in different levels, by BW28878/pUC-Az membranes. Unlike the double His10- AtoCD55G/H73L mutant which was found to be phosphorylated albeit in small levels, which implies that both residues are important for the in vitro phosphorylation of the protein. In the present study was studied, for the first time, the in vitro phosphorylation of AtoC by the cytoplasmic part of AtoS. Autophosphorylation of the His6-cytoAtoS was observed in 30 min time, as was the transfer of the phosphoryl group to then added His10-AtoC. The inclusion of His10-AtoC and the His10-AtoCΗ73L mutant as AtoS substrates showed the elimination of the p-His6-cytoAtoS band with a subsequent appearance of a band responding to His10-AtoC molecular weight. On the contrary, the mutation at Asp55 eliminated the phosphor-tranfer to both D55G and the double mutant. The latter result only reinforces the importance of the conserved Asp55 to the phosphorylation process, a conclusion also idicated by the study of the AtoC ATPase activity. The H-to-L replacement showed no influence on the ATPase activity, contrary to the D-to-G substitution which caused 50% loss of activity, for both His10- AtoCD55G and His10-AtoCD55G/H73L. the ATPase activity was strongly enhanced when the part of the atoDAEB promoter containing both regulation sites was added to the reaction. More specifically, a gel retardation experiment showed that AtoC binds to the ato promoter, and for this binding both regulatory sites are necessary, providing more evidence that AtoC is the transcriptional factor responsible for the induction of the atoDAEB operon by acetoacetate, since the site identified before as the cis-element of the ato promoter responding to acetoacetate also consists the binding region of the response regulator on the promoter. All three AtoC mutants possessed the binding to patoD1 part of the promoter activity at the same levels as the wild-type protein.

Topics: Σύστημα δύο συστατικών, Κινάση ιστιδίνης, Ρυθμιστής απόκρισης, 6s4 μεταγραφικός παράγοντας, Δραστικότητα ατράσης, Φωσφορυλίωση, Two component system, Histidine kinase, Response regulator, 6s4 transcriptional factor, Atrase activity, Phosphorylation
Publisher: Aristotle University Of Thessaloniki (AUTH)
Year: 2006
DOI identifier: 10.12681/eadd/15420
OAI identifier:
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