ATP-Dependent Persister Formation in Escherichia coli

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Physiology

Background:

• Persisters are dormant, antibiotic-tolerant subpopulations of bacteria responsible for chronic infections.
• The prevailing model suggested that guanosine tetraphosphate (ppGpp) activates toxin-antitoxin systems, leading to persister formation.
• The rrnB P1 promoter was proposed as a reporter for persister cells, linked to ppGpp and toxin activation.

Purpose of the Study:

• To investigate the role of toxin-antitoxin systems in persister formation.
• To elucidate the regulatory mechanism underlying the rrnB P1 promoter's activity as a persister marker.
• To identify the primary trigger for bacterial persister formation and antibiotic tolerance.

Main Methods:

• Fluorescence-activated cell sorting (FACS) to analyze cell populations.
• Genetic manipulation, including creating relA/spoT deletion mutants.
• Induction of cellular stress using arsenate to manipulate ATP levels.

Main Results:

• Stress-induced activation of toxin-antitoxin systems did not consistently increase persister numbers.
• The rrnB P1 promoter activity correlates with persister cells but is regulated by ATP levels, not solely by ppGpp or toxins.
• Decreasing cellular ATP levels via arsenate treatment induced drug tolerance and persister formation.
• Lowering ATP levels were shown to reduce antibiotic target activity, including DNA double-strand break formation.

Conclusions:

• Stochastic variation in cellular ATP levels, rather than toxin-antitoxin systems, is the primary mechanism driving persister formation.
• Reduced ATP levels decrease the activity of antibiotic targets, explaining the drug tolerance observed in persister cells.
• The rrnB P1 promoter serves as a reliable indicator of persister cells by responding to ATP fluctuations.