Biochemical determinants of ObgE-mediated persistence.

Autor: Verstraeten N; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Gkekas S; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium.; VIB-VUB Center for Structural Biology, Pleinlaan 2, 1050, Brussels, Belgium., Kint CI; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Deckers B; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium.; VIB-VUB Center for Structural Biology, Pleinlaan 2, 1050, Brussels, Belgium., Van den Bergh B; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Herpels P; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Louwagie E; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Knapen W; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Wilmaerts D; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Dewachter L; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Fauvart M; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; Department of Life Science Technologies, Smart Systems and Emerging Technologies Unit, IMEC, Kapeldreef 75, 3001, Leuven, Belgium., Singh RK; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium.; VIB-VUB Center for Structural Biology, Pleinlaan 2, 1050, Brussels, Belgium., Michiels J; Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium.; VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 20 Box 2460, 3001, Leuven, Belgium., Versées W; Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium.; VIB-VUB Center for Structural Biology, Pleinlaan 2, 1050, Brussels, Belgium.
Jazyk: angličtina
Zdroj: Molecular microbiology [Mol Microbiol] 2019 Nov; Vol. 112 (5), pp. 1593-1608. Date of Electronic Publication: 2019 Sep 18.
DOI: 10.1111/mmi.14382
Abstrakt: 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.
(© 2019 John Wiley & Sons Ltd.)
Databáze: MEDLINE
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