Air-grown cells of the cyanobacterium Synechococcus UTEX 625 were suspended in a cuvette connected to a mass spectrometer and supplied with H13C18O3− to investigate the intracellular interconversion between CO2 and HCO3− as determined from the isotopic composition of CO2 appearing in the extracellular medium under a wide variety of experimental conditions. Upon injection of H13C18O3− to the cell suspension in the light, the extracellular [13C16O2] increased. As the CO2 species were 13C labelled, this demonstrated that the 18O-depleted CO2 was originating from the added H13C18O3−. A comparison of the rates of 13C16O16O appearance in the medium with the formation of 13C16O16O from spontaneous dehydration–hydration in the extracellular medium in the presence of cells demonstrated that most of it had to originate from a series of intracellular dehydration–hydration cycles of H13C18O3− that had been recently transported into the cells. During the time course of the experiments both the m/z (mass to charge) = 49 (i.e., 13C18O18O) and 47 (i.e., 13C18O16O) signals decreased constantly, whereas the m/z = 45 signal (i.e.,13C16O2) always increased. Inhibiting CO2 fixation enhanced the amount of CO2 arising in the medium but did not change its isotopic composition, and the CO2 was always fully depleted of 18O. When the CO2 transport system was inhibited by darkening the cells, adding inhibitors such as Na2S or COS, or quenching the uptake of inorganic 13C with an excess of inorganic 12C, the magnitude of the extracellular [13C16O2] was increased but the CO2 species were still always depleted of 18O. Various incubation times of the illuminated cells in the presence of H13C18O3− were used to obtain a variety of internal Ci pool sizes. When the inhibitor (COS) was added, the amount of 13C16O2 arising during the response time of the mass spectrometer was equivalent to the amount of CO2 that would have been present in the whole cell if CO2 and HCO3− were in equilibrium throughout the entire cell volume, but it was at least 40 times higher than the amount of CO2 that would have been present in the cell if the CO2 was confined to the carboxysomes. Experiments were also conducted at pH 9.0 where the spontaneous rate of 13C16O2 production from H13C1803− dehydration–hydration would be negligible, and again the same features were observed. Results show that intracellular HCO3− and CO2 are in rapid equilibrium throughout the entire cell volume. Key words: Synechococcus UTEX 625, cyanobacteria, CO2 leakage, 18O exchange, active CO2 transport, carboxysomes, inorganic C concentrating mechanism.
The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli
