Time-resolved ATP measurements during vesicle respiration

Area of Science:

• Biochemistry
• Microbiology
• Electrochemistry

Background:

• In vitro adenosine triphosphate (ATP) synthesis by ATP-synthase necessitates membrane vesicles.
• Inverted vesicles from Gram-negative bacteria like Escherichia coli and Pseudomonas putida are suitable for in vitro ATP synthesis studies.
• Current ATP quantification methods, primarily bioluminescence assays, have limitations.

Purpose of the Study:

• To present a novel microbiosensor coupled with scanning electrochemical microscopy (SECM) for ATP level determination.
• To quantify ATP levels in inverted vesicles from E. coli and P. putida using this new method.
• To validate the involvement of the respiratory chain in ATP generation via inhibition studies.

Main Methods:

• Utilized inverted vesicles from E. coli and P. putida.
• Employed a microbiosensor in conjunction with scanning electrochemical microscopy (SECM).
• Developed an innovative two-compartment electrochemical cell for ATP measurement.

Main Results:

• Quantified ATP levels in E. coli vesicles at 0.29 ± 0.03 μM/μl (22 mg/ml protein).
• Quantified ATP levels in P. putida vesicles at 0.48 ± 0.02 μM/μl (25.2 mg/ml protein).
• Venturicidin A inhibition confirmed the role of the respiratory chain in ATP generation.

Conclusions:

• The developed SECM-based microbiosensor provides a sensitive and selective method for ATP quantification in membrane vesicles.
• This technique allows for ATP measurements without sample illumination, overcoming limitations of bioluminescence assays.
• The study confirms the critical role of the respiratory chain in ATP synthesis within these bacterial vesicles.