Sustained net CO2 evolution during photosynthesis by marine microorganisms.

BACKGROUND: Many aquatic photosynthetic microorganisms possess an inorganic-carbon-concentrating mechanism that raises the CO2 concentration at the intracellular carboxylation sites, thus compensating for the relatively low affinity of the carboxylating enzyme for its substrate. In cyanobacteria, the concentrating mechanism involves the energy-dependent influx of inorganic carbon, the accumulation of this carbon--largely in the form of HCO3(-)-in the cytoplasm, and the generation of CO2 at carbonic anhydrase sites in close proximity to the carboxylation sites. RESULTS: During measurements of inorganic carbon fluxes associated with the inorganic-carbon-concentrating mechanism, we observed the surprising fact that several marine photosynthetic microorganisms, including significant contributors to oceanic primary productivity, can serve as a source of CO2 rather than a sink during CO2 fixation. The phycoerythrin-possessing cyanobacterium Synechococcus sp. WH7803 evolved CO2 at a rate that increased with light intensity and attained a value approximately five-fold that for photosynthesis. The external CO2 concentration reached was significantly higher than that predicted for chemical equilibrium between HCO3- and CO2, as confirmed by the rapid decline in the CO2 concentration upon the addition of carbonic anhydrase. Measurements of oxygen exchange between water and CO2, by means of stable isotopes, demonstrated that the evolved CO2 originated from HCO3- taken up and converted intracellularly to CO2 in a light-dependent process. CONCLUSIONS: We report net, sustained CO2 evolution during photosynthesis. The results have implications for energy balance and pH regulation of the cells, for carbon cycling between the cells and the marine environment, and for the observed fractionation of stable carbon isotopes.

Modification of topA in Synechococcus sp. PCC 7942 resulted in mutants capable of growing under low but not high concentration of CO2.

Insertion of a cartridge encoding kanamycin resistance within an open reading frame, ORF839, in the cyanobacterium Synechococcus sp. PCC 7942 resulted in merodiploids bearing both the normal and the modified ORF839, suggesting that its gene product is essential for growth. In the absence of kanamycin the mutants were able to grow like the wild type, but in its presence the mutants grew under 0.015% CO2 in air but not under 5% CO2 in air. ORF839, identified in this study, is highly homologous to topA encoding topoisomerase I in several organisms, but it does not contain the zinc-binding motif identified in the C-terminal region of the enzyme from Escherichia coli.

Evidence for a Na/H Antiporter in Membrane Vesicles Isolated from Roots of the Halophyte Atriplex nummularia.

The ATP-dependent establishment of a positive membrane potential (measured as S(14)CN(-)-accumulation) in membrane vesicles isolated from the roots of Atriplex nummularia Lindl. was not inhibited by NaMes and KMes at concentrations up to 140 millimolar. On the other hand, the formation of DeltapH (measured as (14)C-methylamine accumulation or quenching of quinacrine fluorescence), was depressed by NaMes concentrations as low as 30 millimolar. Supply of NaMes after the DeltapH had been established brought about partial dissipation within 30 seconds. Extent of dissipation of DeltapH increased with NaMes concentration over the range tested (up to 180 millimolar). The H(+)/Na(+) exchange indicated by these results was not due to the creation of a Na(+) diffusion potential. Formation of DeltapH in these vesicles was stable to NO(3) (-) up to 100 millimolar; further, the dissipating effect of Na(+) supply was apparent on a DeltapH formed in the presence of 30 millimolar NO(3) (-). Additional evidence that the origin of the membrane vesicles observed in this investigation was not the tonoplast and was probably the plasmalemma included the vanadate sensitivity of the establishment of the membrane potential.

Generation of a membrane potential by electron transport in plasmalemma-enriched vesicles of cotton and radish.

Plasmalemma-enriched vesicles were isolated from cotton roots (Gossypium hirsutum L. cv Acala San Jose 2) and from germinating radish seeds (Raphanus sativa L. cv Tondo Rosso Quarantino). When 100 millimolar ascorbate was added to the grinding medium, the addition of ferricyanide to either preparation led to an inside positive membrane potential as measured by the accumulation of thiocyanate. It is suggested that electrons from ascorbate were being transported electrogenically across the membrane to ferricyanide, resulting in an accumulation of protons within the vesicle. The redox activity of the vesicles has some similarities to that occurring in intact cells, thus providing a simpler system to study the components and effects of transmembrane electron transport.

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : I. Salinity during Growth Modulates the Proton Pump in the Halophyte Atriplex nummularia.

Membrane vesicles were isolated from the roots of the halophyte Atriplex nummularia Lindl. H(+)-translocating Mg(2+)-ATPase activity was manifested by the establishment of a positive membrane potential (measured as SCN(-) accumulation); and also by the establishment of a transmembrane pH gradient (measured by quinacrine fluorescence quenching). H(+)-translocation was highly specific to ATP and was stable to oligomycin. Growing the plants in the presence of 400 millimolar NaCl doubled the proton-translocating activity per milligram of membrane protein and otherwise modulated it in the following ways. First, the flat pH profile observed in non-salt-grown plants was transformed to one showing a peak at about pH 6.2. Second, the lag effect observed at low ATP concentration in curves relating SCN(-) accumulation to ATP concentration was abolished; the concave curvature shown in the double reciprocal plot was diminished. Third, sensitivity to K-2 (N-morpholino)ethanesulfonic acid stimulation was shown in salt-grown plants (about 40% stimulation) but was absent in non-salt-grown plants. Fourth, the KCl concentration bringing about 50% dissipation of ATP-dependent SCN(-) accumulation was 20 millimolar for salt-grown plants and 50 millimolar for non-salt-grown plants. Vanadate sensitivity was shown in both cases. No clear NO(3) (-) inhibition was observed.

Studies on H-Translocating ATPases in Plants of Varying Resistance to Salinity : II. K Strongly Promotes Development of Membrane Potential in Vesicles from Cotton Roots.

Mg(2+)-ATP-dependent H(+)-translocation has been studied in membrane vesicles derived from the roots of Gossypium hirsutum L. var. Acala San Jose 2. Establishment of a positive membrane potential was followed by measuring SCN(-) accumulation; establishment of DeltapH across the vesicle membranes by measuring quinacrine fluorescence quenching. High specificity for ATP was shown, and H(+)-translocation was oligomycin stable. The pH profile for H(+)-translocation showed an optimum at 5.5. The relationship between SCN(-) accumulation and ATP concentration was approximately Michaelian; the apparent K(m) was 0.7 millimolar. K-2-(N-morpholino)ethanesulfonic acid strongly promoted ATP-dependent SCN(-) uptake (up to 180% stimulation). The effect was not given by Na-Mes. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone totally inhibited SCN(-) accumulation, both in the presence and absence of K-2(N-morpholino)ethanesulfonic acid. Vanadate at 200 micromolar inhibited SCN(-) uptake by about 10 to 40% in the absence of K(+), but more strongly in its presence (about 60%). NO(3) (-) at 100 millimolar inhibited initial rate of quinacrine quenching by about 25%. The NO(3) (-) insensitive fraction was activated by K(+); and inhibited by 200 micromolar vanadate to about 40%, provided K(+) was present. Saline conditions during the growth of the plants had no appreciable effect on the observed characteristics of H(+)-translocation.

Phenotypic Complementation of High CO(2)-Requiring Mutants of the Cyanobacterium Synechococcus sp. Strain PCC 7942 by Inosine 5'-Monophosphate.

The high CO(2)-requiring mutants of Synechococcus PCC 7942, D4 and R14, were obtained by deletion or inactivation (respectively) of an open reading frame immediately downstream of rbc (the operon encoding the subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase). These mutants exhibit photosynthetic characteristics similar to those of high CO(2)-grown wild type, unlike other cyanobacterial high CO(2)-requiring mutants, where the apparent photosynthetic affinity for inorganic carbon is approximately 2 orders of magnitude lower than that of the wild type. Sequence analysis and metabolic complementation of the mutants by inosine 5'-monophosphate identified this open reading frame as the cyanobacterial equivalent of purK, the eubacterial gene encoding subunit II of phosphoribosyl aminoimidazole carboxylase in the purine biosynthetic pathway. Exposure of high CO(2)-grown Synechococcus to low CO(2) conditions led to the induction of transcription of purK. It is suggested that the high CO(2)-requiring phenotype of these mutants resulted from the defect in purine biosynthesis after exposure to low CO(2). We also raise the possibility that the level of cellular purines is involved in the process of adaptation of cyanobacteria to low concentrations of CO(2).

Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton.

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the DeltapH probe [(14)C]methylamine. Evidence is presented for the operation of Na(+)/H(+) and K(+)/H(+) antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H(+)-ATPase or by imposing an artificial pH gradient) brings about dissipation of the DeltapH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl(-), using the DeltaPsi probe SCN(-). Cation/H(+) exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H(+)-ATPase; coupling of the opposed cation and H(+) fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na(+)/H(+) and K(+)/H(+) antiport in Atriplex. Moreover, additive effects are obtained when Na(+) is supplied together with saturating concentrations of K(+), and vice versa, suggesting that separate antiporters for Na(+) and for K(+) may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.

A Cyanobacterial Gene Encoding Peptidyl-Prolyl cis-trans Isomerase.

The rotA gene encoding peptidyl-prolyl cis-trans isomerase has been identified, sequenced, and shown to be transcribed in the cyanobacterium Synechococcus sp. PCC7942. Inactivation of the gene by replacement of a region containing the open reading frame with a gene conferring kanamycin resistance resulted in merodiploids containing both the wild type and the modified genomic region. We were not able to isolate a kanamycin-resistant mutant in which all the genomic wild-type copies were substituted, which suggests that such replacement could have been lethal.