Biochemical determinants of ObgE-mediated persistence

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Obg GTPases are crucial for bacterial persistence.
• ObgE in Escherichia coli activates toxin-antitoxin modules and causes membrane depolarization.
• The G-domain's role in ObgE's persistence function was previously unclear.

Purpose of the Study:

• To investigate the role of G-domain functionality in ObgE-mediated bacterial persistence.
• To identify specific mutations affecting ObgE's persistence function.
• To correlate persistence function with nucleotide binding and GTPase activity.

Main Methods:

• Screening of an obgE mutant library.
• Site-directed mutagenesis based on ObgE crystal structure.
• Isothermal titration calorimetry, stopped-flow experiments, and kinetic assays.
• Assessment of hokB expression and bacterial viability.

Main Results:

• Five obgE alleles (G166V, D246G, S270I, N283I, I313N) lost persistence function but retained essential roles.
• Designed mutants T193A and D286Y further supported the uncoupling of persistence and essential functions.
• Mutants lacking persistence showed significantly reduced binding to GDP and ppGpp, but not GTP or pppGpp.
• No clear correlation was found between persistence and GTP hydrolysis activity.

Conclusions:

• ObgE's persistence function can be separated from its essential cellular roles.
• GDP and ppGpp binding are critical for ObgE's persistence activity.
• Understanding these biochemical determinants is key to deciphering bacterial persistence mechanisms.