The rate of accumulation of streptomycin by streptomycin-sensitive strains of Escherichia coli and Bacillus megaterium, grown in chemostats, was related to the growth rate prior to addition of the antibiotic. For E. coli the length of the lag period that preceded accumulation was also growth rate-dependent. Thus faster growing cultures accumulated streptomycin more rapidly and with a shorter lag than slower growing cultures. The rate of efflux of streptomycin from bacteria that had accumulated streptomycin was not greatly influenced by growth rates of the cultures. At a particular growth rate, accumulation of streptomycin was found to be faster at higher concentrations of the antibiotic. Rapid accumulation of streptomycin was not observed with continuous cultures of a streptomycin-resistant strain of E. coli. Accumulation of streptomycin was abolished when growth was inhibited by either terminating the flow of fresh medium to a chemostat or by adding inhibitors that block protein synthesis. These results suggest that the rate of accumulation of streptomycin is related to the concentration of streptomycin-sensitive ribosomes that are actively engaged in protein synthesis within the bacterial cells.
The growth yields of Escherichia coli on glucose, lactose, galactose, maltose, maltotriose, and maltohexaose were estimated under anaeorbic conditions in the absence of electron acceptors. The yields on these substrates exhibited significant differences when measured in carbon-limited chemostats at similar growth rates and compared in terms of grams (dry weight) of cells produced per mole of hexose utilized. Maltohexaose was the most efficiently utilized substrate, and galactose was the least efficiently utilized under these conditions. All these sugars were known to be metabolized to glucose 6-phosphate and produced the same pattern of fermentation products. The differences in growth yields were ascribed to differences in energy costs for transport and phosphorylation of these sugars. A formalized treatment of these factors in determining growth yields was established and used to obtain values for the cost of transport and hence the energy-coupling stoichiometries for the transport of substrates via proton symport and binding-protein-dependent mechanisms in vivo. By this approach, the proton-lactose stoichiometry was found to be 1.1 to 1.8 H+ per lactose, equivalent to approximately 0.5 ATP used per lactose transported. The cost of transporting maltose via a binding-proteindependent mechanism was considerably higher, being over 1 to 1.2 ATP per maltose or maltodextrin transported. The formalized treatment also permitted estimation of the net ATP yield from the metabolism of these sugars; it was calculated that the growth yield data were consistent with the production of 2.8 to 3.2 ATP in the metabolism of glucose 6-phosphate to fermentation products.
Using chemostat cultures of Escherichia coli it was possible to vary respiration rates while maintaining a constant growth rate. This allowed the effect of variations in respiration rates on the accumulation of streptomycin to be studied in cultures at constant growth rates. At a particular dilution rate cultures exhibited higher respiration rates when phosphate limited growth than when carbon limited growth. A ubiquinone-deficient strain had a lower rate of respiration at a particular dilution rate than a related ubiquinone-sufficient strain. In spite of these differences in respiratory activity, the accumulation of streptomycin was identical in carbon-and in phosphate-limited chemostat cultures of ubiquinone-deficient and ubiquinonesufficient strains. Moreover, accumulation of streptomycin in an anaerobic chemostat culture occurred at the same rate as that in an aerobic chemostat. There was however a lag of 1.5 h before accumulation commenced in the anaerobic culture, a feature that was not apparent in the aerobic culture. These results indicate that the lower rates of respiration in slow-growing bacteria are not responsible for the decreased accumulation of streptomycin in slow-growing compared to fast-growing cultures. Moreover, it seems unlikely that quinones are involved directly (e.g. as carriers) in streptomycin accumulation, since removal of 90% of cellular ubiquinone, or replacement of ubiquinone with a structural analogue, did not affect accumulation as long as mutant and parent cultures grew at the same rate.
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