Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis

Abstract

The nature of the inorganic carbon (C,) species actively taken up by cyanobacteria CO2 or HC03-has been investigated. The kinetics of CO2 uptake, as well as that of HC03-uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO2 was higher than that for HCO3-. Though the calculated V. was the same in both cases, the maximum rate of uptake actually observed was higher when HC03-was supplied. C. uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO2 was the species supplied. Observations of photosynthetic rate as a function of intracellular C. level (following supply of CO2 or HCO3-for 5 seconds) led to the inference that HC03-is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C. transport apparatus binds CO2 and releases HC03-at or near a membrane porter. The latter transports HC03-ions to the cell interior.The observed capability of green algae and cyanobacteria to accumulate Ci2 within the cells has been attributed to the operation of a mechanism for active transport of HCO3 ions (for review, see Lucas [8]). However, the observation that supply of '4C02 to green algae (5, 9, 1 1) and cyanobacteria (2) leads to faster accumulation of C. and acid-stable '4C than does HCO3 supply has prompted the proposal that CO2 may in fact be the species actively transported. In the present study we have investigated the uptake of each C, species within a brief period of its supply to Anabaena variabilis cells and, further, have examined interactions between CO2 and HCO3 during the uptake process. Data obtained bear on the molecular mechanism involved in C. uptake in cyanobacteria. MATERIALS AND METHODSCultures of Anabaena variabilis M-3 (from the collection of Tokyo University) were grown as described earlier (6). Accumulation of acid stable (photosynthetic products) and labile (inorganic) carbon were determined using the filtering centrifugation technique (6, 7 injected into closed microfuge tubes containing 100 mm Mes (pH 2.4). Following this injection the pH rose to 5.1 (maximum). The solution in the tube was mixed for 60 s with a syringe and the required volume withdrawn and injected into the cell suspension. Another sample was injected into 0.1 N NaOH. The radioactivity in the letter sample was assessed in order to correct for the loss of 14C02 from the microfuge tube. This procedure enabled accurate determination of the concentration of CO2 supplied, and ensured that the specific activity of the C. supplied was the same, whether presented as CO2 or HCO3 . The injection of aliquots of the H'4CO3 or '4CO2 stocks changed the pH of the cell suspension (in 50 mM Hepes-NaOH, pH 8.0) to only a minor degree (maximum 0.1 pH units). This change was taken into account whenever the actual...

Selective and Reversible Inhibition of Active CO2 Transport by Hydrogen Sulfide in a Cyanobacterium

“…To this end, several groups have reported that the CA inhibitor EZ inhibits CO2 transport in Anabaena variabilis (1,28). However, the results ofa recent study by Price and Badger (25) using Synechococcus PCC 7942 suggests that EZ, at 200 gM, equally inhibits CO2 and HCO3-transport.…”

“…Previous studies have suggested the participation ofa membrane-bound CA-like moiety in the transport of CO2 by Anabaena variabilis, Anacystis nidulans R2, and Synechococcus PCC 7942 (1,22,25,28). This conclusion was largely based on the observation that EZ inhibited CO2 transport (1,22,25,28) without apparent inhibition of intracellular CA (25).…”

“…The difference in peak height between experimental curves (+ Na2S) and the curve obtained in the dark was was made to calculate rates of CO2 transport (17). Nevertheless, the method provides an alternate means of assessing CO2 transport, as distinct from HCO3-transport, over a range of [CO2] and is the procedure most directly comparable to 4GCO2 pulsing techniques used in conjunction with silicone fluid filtering centrifugation (2,17,28) and isotopic disequilibrium experiments (6).…”

“…During steady state photosynthesis in Synechococcus leopoliensis UTEX 625 both species of DIC are transported simultaneously and continuously in light dependent processes (7). Although cyanobacteria rapidly remove CO2 from the medium (2,7,17,28), it is HCO3-transport which provides the bulk of the DIC to the intracellular pool when the extracellular pH is alkaline and the [DIC] low (2,6,7,10,16 …”

“…Cyanobacteria possess mechanisms for the active transport of CO2 and HC03-that lead to the accumulation of a large intracellular pool of DIC3 (2,6,10,11,13,16,17,28) which serves as the immediate source of CO2 for photosynthesis (10,16). During steady state photosynthesis in Synechococcus leopoliensis UTEX 625 both species of DIC are transported simultaneously and continuously in light dependent processes (7).…”