The flow of ions has long been appreciated as a means of cellular signaling. While well characterized in electrically excitable eukaryotic cells, electrophysiological studies in bacteria were limited by a lack of tools that function in living cells. Bacterial electrophysiology influences metabolism, division, and persistence through antibiotic treatment, but little is known about its role in signaling. The development of novel fluorescent voltage indicators revealed that bacteria contain rapid membrane depolarization and repolarization events. My project strives to uncover the physiological role of these voltage transients in E. coli. Using a combination of fluorescent voltage and calcium biosensors, we found bacteria are similar to electrically excitable eukaryotic cells; voltage depolarization induces calcium influx in individual E. coli and S. enterica Typhimurium. We discovered that cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent upon the same stimulus. Blocking electrophysiology flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. This evidence suggests that voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. In order to understand downstream consequences of altered electrophysiology we have completed a small molecule screen by combining semi-automated microscopy with GCaMP6f expressing E. coli and found compounds that alter electrophysiology in E. coli. We have used these compounds to explore how electrophysiology influences S. enterica Typhimurium infection of HeLa cells; compounds that increase bacterial electrophysiological flux increased the amount of infected human cells, and vice versa. Finally utilizing the genetically encoded indicators mentioned above, we have evidence of the mechanism of aminoglycoside induced cell death in E. coli. After aminoglycoside treatment, ribosome instability causes ATP accumulation that runs the F1F0-ATPase in reverse, which in turn causes membrane hyperpolarization. Those cells that hyperpolarize are unable to recover from the aminoglycoside treatment. These data provide evidence that dynamic electrophysiology exists as a signaling modality in single celled organisms and can be manipulated to change their lifestyle.
|Advisor:||Kralj, Joel M|
|Commitee:||Stowell, Michael, Palmer, Amy, Detweiler, Corrie, Niswander, Lee|
|School:||University of Colorado at Boulder|
|Department:||Molecular, Cellular and Developmental Biology|
|School Location:||United States -- Colorado|
|Source:||DAI 81/11(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biophysics, Microbiology|
|Keywords:||Antibiotic, Bacteria, Electrophysiology, Microscopy, Rhodopsin, Voltage|
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