Measurement of the amount and isotopic composition of the CO2released from the cyanobacteriumSynechococcusUTEX 625 after rapid quenching of the active CO2transport system

Abstract

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 CO2and HCO3−as determined from the isotopic composition of CO2appearing 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 CO2species were13C labelled, this demonstrated that the18O-depleted CO2was originating from the added H13C18O3−. A comparison of the rates of13C16O16O appearance in the medium with the formation of13C16O16O 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 CO2fixation enhanced the amount of CO2arising in the medium but did not change its isotopic composition, and the CO2was always fully depleted of18O. When the CO2transport system was inhibited by darkening the cells, adding inhibitors such as Na2S or COS, or quenching the uptake of inorganic13C with an excess of inorganic12C, the magnitude of the extracellular [13C16O2] was increased but the CO2species were still always depleted of18O. Various incubation times of the illuminated cells in the presence of H13C18O3−were used to obtain a variety of internal Cipool sizes. When the inhibitor (COS) was added, the amount of13C16O2arising during the response time of the mass spectrometer was equivalent to the amount of CO2that would have been present in the whole cell if CO2and HCO3−were in equilibrium throughout the entire cell volume, but it was at least 40 times higher than the amount of CO2that would have been present in the cell if the CO2was confined to the carboxysomes. Experiments were also conducted at pH 9.0 where the spontaneous rate of13C16O2production from H13C1803−dehydration–hydration would be negligible, and again the same features were observed. Results show that intracellular HCO3−and CO2are in rapid equilibrium throughout the entire cell volume. Key words: Synechococcus UTEX 625, cyanobacteria, CO2leakage,18O exchange, active CO2transport, carboxysomes, inorganic C concentrating mechanism.