Porins are transmembrane proteins within the outer membrane of gram-negative bacteria that allow nutrients, and small molecules, including antibiotics, to enter the cell. Electrophysiological studies on porins reconstituted in lipid bilayers have suggested a dynamic regulation of porin conductance through changes in the ionic environment. Thus, this thesis focuses on understanding porin permeability regulation in live bacteria, employing Escherichia coli as a model organism. Firstly, using a flow cytometric method that I developed, I showed that the uptake of a fluorescent glucose analogue, and other porin-dependent compounds, was modulated by changes in the internal H+ and K+ levels (achieved using ionophores and different external solutions). I then exploited an optically activated proton pump to increase periplasmic H+ and demonstrate that this reduced porin permeability (monitored by live single-cell imaging). In support of a model dependent on changes in periplasmic ions, molecular dynamic simulations suggest that the pore diameter of porins is predicted to be reduced by increased periplasmic H+ ions. Secondly, I examined the dynamic regulation of internal ions in live bacteria by expressing a wide range of genetically encoded fluorescent ion sensors in cells trapped in a microfluidic device. I found that the periplasmic ionic environment is partially insulated from the external ion concentrations and observed oscillations in periplasmic and cytosolic H+ and cytosolic K+ ions. I observed that inner membrane voltage is principally controlled by H+ and K+ gradients and is characterised by spikes (or action potentials) driven by activation of the voltage-gated potassium channel Kch, which acts to increase periplasmic H+ and K+ levels and, as a consequence, increases porin permeability. By examining ion changes and porin permeability of bacteria under starvation or in minimal media supplemented with low or high glucose levels or lipids, I developed a model for metabolic control of porin function. Under starvation (with predicted low levels of periplasmic H+ and K+), porins are open. Porins shut when bacteria are exposed to low glucose or lipid media (where periplasmic H+, but not K+ levels rise). However, under conditions of high metabolism, porins open again (since Kch is activated, leading to low H+ and high K+ levels in the periplasm). Thirdly, I tested my model's predictions and clinical relevance by examining the permeability and susceptibility of ciprofloxacin (an antibiotic known to rely on porins for entry into bacteria). As expected, uptake and subsequent activity of ciprofloxacin was significantly lower in lipid compared to glucose media and could be increased by increasing bacterial H+ levels. Ciprofloxacin uptake was higher in wild type bacteria cells exposed to high rather than low glucose, and this phenomenon relies on Kch activity. My work demonstrates dynamic regulation of porin permeability, which will have important clinical implications for antibiotic development and for potentially explaining the increase in antibiotic resistance seen in lipid media (and intracellular bacteria) and the development of deleterious mutations in central metabolism genes during adaptation to antibiotic treatment. Finally, I examine the biological impact of the only intrinsically produced protonophore, indole. I describe two patterns of indole production depending on the carbon source: glycolytic sugars trigger a strong indole pulse, while gluconeogenic carbon sources result in steady indole production. These behaviours are mainly controlled by modulating the synthesis of tryptophanase, which is responsible for indole production. I show that indole production modulates the membrane potential, suggesting a potential role in regulating porin permeability and other bacterial processes. The work presented here constitutes a great leap forward in our understanding of the role and functions of the periplasmic space. Future research could use our results for the development of adjuvant molecules that take advantage of our framework to increase permeability to antibiotics.