Biochemical determinants of ObgE-mediated persistence.

TitleBiochemical determinants of ObgE-mediated persistence.
Publication TypeJournal Article
Year of Publication2019
AuthorsVerstraeten, N., S. Gkekas, C. Ines Kint, B. Deckers, B. Van den Bergh, P. Herpels, E. Louwagie, W. Knapen, D. Wilmaerts, L. Dewachter, M. Fauvart, R. Kumar Singh, J. Michiels, and W. Versées
JournalMol Microbiol
Volume112
Issue5
Pagination1593-1608
Date Published2019 11
ISSN1365-2958
Abstract

Obg is a versatile GTPase that plays a pivotal role in bacterial persistence. We previously showed that the Escherichia coli homolog ObgE exerts this activity through transcriptional activation of a toxin-antitoxin module and subsequent membrane depolarization. Here, we assessed the role of G-domain functionality in ObgE-mediated persistence. Through screening of a mutant library, we identified five obgE alleles (with substitutions G166V, D246G, S270I, N283I and I313N) that have lost their persistence function and no longer activate hokB expression. These alleles support viability of a strain otherwise deprived of ObgE, indicating that ObgE's persistence function can be uncoupled from its essential role. Based on the ObgE crystal structure, we designed two additional mutant proteins (T193A and D286Y), one of which (D286Y) no longer affects persistence. Using isothermal titration calorimetry, stopped-flow experiments and kinetics, we subsequently assessed nucleotide binding and GTPase activity in all mutants. With the exception of the S270I mutant that is possibly affected in protein-protein interactions, all mutants that have lost their persistence function display severely reduced binding to GDP or the alarmone ppGpp. However, we find no clear relation between persistence and GTP or pppGpp binding nor with GTP hydrolysis. Combined, our results signify an important step toward understanding biochemical determinants underlying persistence.

DOI10.1111/mmi.14382
Alternate JournalMol. Microbiol.
PubMed ID31498933
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