Bacterial membrane potential is difficult to measure using classical electrophysiology techniques due to the small cell size and the presence of the peptidoglycan cell wall. Instead, chemical probes are often used to study membrane potential changes in conditions of interest. Many of these probes are fluorescent molecules that accumulate in a charge-dependent manner, and the resulting fluorescence change can be analyzed via flow cytometry or using a fluorescence microplate reader. Although this technique works well in many Gram-positive bacteria, it generates fairly low signal-to-noise in Gram-negative bacteria due to dye exclusion by the outer membrane. We detail an optimized workflow that uses the membrane potential probe, DiOC2 (3), to measure E. coli membrane potential changes in high throughput and describe the assay conditions that generate significant signal-to-noise to detect membrane potential changes using a fluorescence microplate reader. A valinomycin calibration curve demonstrates this approach can robustly report membrane potentials over at least a ∼144 mV range with an accuracy of ∼12 mV. As proof of concept, we used this approach to characterize the effects of some commercially available small molecules known to elicit membrane potential changes in other systems, increasing the repertoire of compounds known to perturb E. coli membrane energetics. One compound, the eukaryotic Ca2+ channel blocker amlodipine, was found to alter E. coli membrane potential and decrease the MIC of kanamycin, further supporting the value in this approach of screening. This detailed methodology permits studying E. coli membrane potential changes quickly and reliably at the population level.

1. The interrelationship of metabolic activities in chloroplasts and cytoplasm of leaf cells of spinach, sugar beet and Elodea has been investigated. Different methods have been adopted to study the intracellular localization of enzymes and the flow of phosphorylated intermediates across the chloroplast membrane. The flow of substrates was investigated by determining the rates of the conversion of substrates added to aqueously isolated chloroplasts, prior to and after destruction of the outer chloroplast membrane. The observed differences yielded information as to whether a substrate could traverse the chloroplast membrane.Two methods mere used to investigate the localization of enzymes :a) The percentage distribution of photosynthetic and respiratory enzymes in chloroplasts and cytoplasm was calculated from data on enzyme activities in non-aqueous cell fractions.b) Low levels of enzymes in chloroplasts in the presence of high cytoplasmatic levels were detected by assaying enzyme activities in preparations of aqueously isolated chloroplasts prior to and after ultrasonic destruction of the outer chloroplast membrane.2. If chloroplasts are isolated in aqueous sucrose buffer, their outer membranes act as an efficient barrier against the penetration of NADP, RuDP, GAP and, in some but not all experiments, of FMP and GMP. PGA, DHAP and, probably to a lesser extent, aspartate, ɑ-ketoglutarate, oxaloacetate and FDP can traverse this membrane. Chloroplast membranes are significantly altered when isolated in NaCI-buffer systems and do not correspond to the in vivo situation.3. The conversion of Ri-5-P to RuDP occurs exclusively or nearly exclusively in the chloroplasts indicating that phosphoribulokinase and/or ribosephosphate isomerase are located only there.4. The conversion of Ri-5-P to GAP and SuMP, which is catalyzed by the enzymes ribosephosphate isomerase, xylulosephosphate epimerase and transketolase, proceeds likewise only or at least predominantly in the chloroplasts and not, or only to a small extent, in the cytoplasm.5. The major parts of glucose-6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase reside in the cytoplasm. However, a small, but significant, level of these enzymes is to be found also in the chloroplasts. Hexokinase and transaldolase are also present there. Pyruvate kinase and phosphofructokinase appear to be absent from chloroplasts.6. Since, with the presence of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, transaldolase and enzymes of the Calvin cycle, the enzymic machinery of the oxidative pentose phosphate pathway is complete in the chloroplasts, the results suggest that chloroplasts are engaged in the oxidative decomposition of carbohydrates.7. In the dark the oxidative pentose phosphate pathway requires the control of NADPH formation and the transfer of hydrogen across the chloroplast membrane.8. The available data on the intracellular localization of enzymes and on the kinetics of the distribution of labelled intermediates show that the photosynthetic carbon cycle operates exclusively within the chloroplasts. There is nothing to suggest that enzymes of chloroplasts and cytoplasm cooperate in the cyclic regeneration of the carbon acceptor molecule. However, the existence of phosphorylated transport metabolites suggests that secondary reactions of photosynthesis such as sucrose and amino acid synthesis, which proceed, at least in part, outside the chloroplasts, are directly linked with chloroplastic reactions by activated (phosphorylated) intermediates.

TrkH is a bacterial ion channel implicated in K + uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.

The distribution of phosphoglyceric acid (PGA) * between chloroplasts and cytoplasm of leaf cells during transients from dark to light and vice versa has been investigated. The data indicate that pools of PGA in the chloroplasts and cytoplasm are interchangeable and that PGA may function as a transport metabolite in actively metabolising leaf cells. These views are supported by the following results:1. In the presence of 14CO2 and light, labelled PGA rapidly appears in the cytoplasm, even though the carboxydismutase reaction, in which 14C enters into PGA, proceeds in the chloroplasts.2. After less than 1 min. illumination in the presence of 14CO2, the distribution of labelled PGA between chloroplasts and cytoplasm reaches an equilibrium, which is then maintained. The same distribution is to be found by enzymatic analyses of the total pools of PGA in chloroplasts and cytoplasm. When equilibrium is reached, the percentages of both 14C labelled and of total PGA to be found in the chloroplasts of Spinach and Elodea are approximately 75% and 35 —40% respectively.3. In both the chloroplasts and the cytoplasm, the levels of PGA first decrease after illumination to a fraction of the original dark levels and then show a concomitant slow increase. On darkening a further very rapid increase in PGA occurs in chloroplasts and cytoplasm.4. In photosynthetically active leaf material the rate of decrease in the level of cytoplasmic PGA, as observed after 12 —15 secs. illumination, is higher than the turnover rate of PGA in respiration.5. Upon illumination, aqueously isolated chloroplasts, suspended in isotonic sucrose buffer, reduce added PGA to dihydroxyacetone phosphate and other products far faster than they reduce added NADP. Whereas PGA reduction is not increased by ultrasonic disintegration of the chloroplasts, the reduction of NADP is stimulated. This indicates that whereas the movement of NADP is prevented by a permeability barrier, the transferance of PGA across the chloroplast membrane occurs easily.6. In illuminated Elodea shoots the inhibition of metabolism by cyanide after 15 secs. photosynthesis in the presence of Η14CO3⊖ leads to a rapid decrease in PGA. This applies to both the 14C labelled PGA and the total PGA to a similar extent. The decrease in PGA amounts from 70 — 85% of the original dark levels. Since the chloroplasts of Elodea contain only 35—40% of the total PGA of the cell, a fall in the level of PGA as a result of the cyanide poisoning obviously occurs not only in the chloroplasts, but also in the cytoplasm. Since cyanide effectively inhibits cytochrome oxidase, while PGA reduction in the chloroplasts is relatively resistant, the large decrease in PGA suggests that part of the cytoplasmic PGA is transferred into the chloroplasts and reduced there.