Probing Escherichia coli Energetics under Starvation by Single-Cell Measurements

• Main Authors: Ssu-Yuan Lin, 林思遠
• Other Authors: Chien-Jung Lo
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/zfxth2

碩士 === 國立中央大學 === 物理學系 === 106 === ‘’How much energy does a bacteria cell require to survive?’’ ‘’Is there a minimum energy requirement for a bacterial cell to maintain viability?’’ Energy is crucial to life. A simplistic view of life is to think of it as a process that transforms external materials into cellular components based on genetic information and by transducing energy. Energy is required for all of the biological processes. While most of the efforts by biologists temp to understand the flow of genetic information, very little is known regarding cellular energetic flow. Furthermore, the crisis of superbugs, inspire us to focus on the fundamental understanding of bacterial energetic for alternative route of antibiotic discovery. Therefore, we conduct single-cell energetic experiments to probe the dynamics and boundary between life and death of bacteria under extreme starvation. There are two major types of energy sources in bacteria, adenosine triphosphate (ATP) and pronton motive force (PMF). ATP is a universal energy currency in all forms of life. PMF is the combination of electrical and chemical potential difference across bacterial cell membrane. In this thesis, we use the fluorescent protein (QUEEN-QUantitative Evaluator of cellular ENergy)[1] and the bacterial flagellar motor (BFM) to probe the ATP and PMF respectively. QUEEN sensor is a circularly-permuted ATP sensitive green fluorescent protein. BFM is the molecular motor driven by ion flux and PMF. By these two cutting-edge biophysical probes, we could measure the dynamics of these two main energy source in a bacteria cell. We aim to measure the ‘free energy’ of a bacterial cell could utilize under extreme starvation. Besides, we also want to know if we could starve a bacterial cell to death. After Escherichia coli cells being transferred from rich medium to zero nutrient medium, the BFM rotation speed drops exponentially with a load-dependent decay rate. The total energy consumed through BFM while starvation is roughly on the scale of mM ATP, equivalent to 10-13 Joule per E. coli and is load–independent. After BFM speed and PMF drop to zero, the cells are retreated with rich medium. Surprisingly, most bacterial cells are viable and can recover from the starvation. The single cell measurement of intracellular ATP by QUEEN fluorescent protein shows the same order of energy before starvation. The measurements shed a new way to probe bacterial energetics and dynamical response under starvation.