mrpA, a gene with roles in resistance to Na+ and adaptation to alkaline pH in the cyanobacterium Anabaena sp. PCC7120.

Transposon mutagenesis of Anabaena sp. PCC7120 led to the isolation of a mutant strain, PHB11, which grew poorly at pH values above 10. The mutant strain exhibited pronounced Na+ sensitivity; this sensitivity was higher under basic conditions. Mutant PHB11 also showed an inhibition of photosynthesis that was much more pronounced at alkaline pH. Reconstruction of the transposon mutation of PHB11 in the wild-type strain reproduced the phenotype of the original mutant. The wild-type version of the mutated gene was cloned and the mutation complemented. In mutant strain PHB11, the transposon had inserted within an ORF that is part of a seven-ORF operon with significant sequence similarity to a family of bacterial operons that are believed to code for a novel multiprotein cation/proton antiporter primarily involved in resistance to salt stress and adaptation to alkaline pH. The Anabaena operon was denoted mrp (multiple resistance and pH adaptation) following the nomenclature of the Bacillus subtilis operon; the ORF mutated in PHB11 corresponded to mrpA. Computer analysis suggested that all seven predicted Anabaena Mrp proteins were highly hydrophobic with several transmembrane domains; in fact, the predicted protein sequences encoded by mrpA, mrpB and mrpC showed significant similarity to hydrophobic subunits of the proton pumping NADH : ubiquinone oxidoreductase. In vivo expression studies indicated that mrpA is induced with increasing external Na+ concentrations and alkaline pH; mrpA is also upregulated under inorganic carbon (Ci) limitation. The biological significance of a putative cyanobacterial Mrp complex is discussed.

Changes in carboxysome structure and grouping and in photosynthetic affinity for inorganic carbon in Anabaena strain PCC 7119 (Cyanophyta) in response to modification of CO2 and Na+ supply.

In ANABAENA: PCC 7119 a 4-fold decrease in the value of the apparent photosynthetic affinity for external inorganic carbon [K1/2 (Ci)] occurred between 9 and 12 h after the transfer from high-CO2 (2% CO2-enriched air) to air-growing conditions. A slight increase in carboxysome frequency occurred, but during this transition their appearance and distribution remained unchanged. ANABAENA: PCC 7119 did not improve its K1/2 (Ci) beyond the above cited level of acclimation neither by culturing the cyanobacteria in Na+-deficient medium in air nor by aeration with CO2-depleted air. In air-grown cultures, Na+ deficiency induced a large increase in carboxysome frequency and an alteration of their appearance: the greatest proportion were electron-dense whereas this type constituted a minority in high-CO2 and in air, Na+-sufficient conditions. It also induced major changes in carboxysome distribution, whereby more than 60% were grouped, compared with only 10% in high-CO2 and in air, Na+-sufficient conditions. These changes in carboxysome expression were extremely rapid, occurring mainly during the first 2 h.

pbpB, a gene coding for a putative penicillin-binding protein, is required for aerobic nitrogen fixation in the cyanobacterium Anabaena sp. strain PCC7120.

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i. e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N(2), are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

N2-Fixation in Cyanobacterial Mats from Ponds on the McMurdo Ice Shelf, Antarctica.

We have investigated the ecological importance of N2-fixation in cyanobacterial mats, dominated by oscillatorean species, in ponds of the Bratina Island area of the McMurdo Ice Shelf, Antarctica (78 degrees S, 166 degrees E). Nitrogenase activity, estimated as acetylene reducing activity (ARA), was found in all the mats investigated (n = 16). The average ARA was 75.9 mmol ethylene m-2 h-1, ranging from 6 to 201 mmol ethylene m-2 h-1. Nitrogenase activity was positively correlated with dissolved reactive phosphorus concentration in pondwater and the C/N ratio of the mat, and was negatively correlated with pondwater NH4+-N concentrations and natural abundance of 15N in the mats. ARA was restricted to the upper, oxic layer of the mats. Experiments conducted to ascribe ARA to different groups of prokaryotes suggested that ARA was mainly conducted by heterocystous cyanobacteria, since no activity was found in the dark and the activity was inhibited by the photosystem II inhibitor DCMU (3-[3,4-dichlorophenyl]-1,1-dimethyl urea). In spite of 24 h of daylight, nitrogenase activity showed a diel cycle with maximum activity at midday (10-18 h) and minimal activity at early morning (6-10 h) when pond temperatures were at their minima. Light dependency of nitrogenase activity for three cyanobacterial communities showed that the irradiance required for saturating ARA was low, in every case lower than 100 mmol photon m-2s-1. Irradiance rarely fell below 100 mmol photon m-2s-1 during Antarctic summer days and ARA was likely to be light saturated for much of the time. We estimate that N2 fixation represented on average a N input into the ponds of over 1 g m-2y-1. This value appears to be the highest N input to this Antarctic ecosystem.

Effects of phenoxy acetic herbicides on growth, photosynthesis, and nitrogenase activity in cyanobacteria from rice fields.

The effects of the phenoxy acetic herbicides 2,4-dichlorophenoxy acetic acid (2,4D) and methylchlorophenoxy acetic acid (MCPA) on growth, photosynthesis, and nitrogenase activity of cyanobacteria has been investigated. Concentrations ranging from 10(-9) to 10(-3) M did not change significantly the parameters of Anabaena UAM 202. Concentrations higher than 10(-3) M of both herbicides were toxic. The primary toxic action of these herbicides in Anabaena UAM 202 was on photosynthesis.

Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119.

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N(2) fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O(2) evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N(2) fixation in cyanobacteria.

Protection by CO(2) against Sodium Deficiency in a Mutant of Nostoc muscorum.

The effects of Na-deficient culture were studied on a mutant of Nostoc muscorum unable to fix N(2). Na deficiency made the cells sensitive to photooxidation, thus at a light intensity of lO W m(-2) and a low concentration of CO(2) (0.03 %), Na deficiency caused chlorosis and cell lysis. At a lower light intensity, cell damages did not take place. Higher levels of CO(2) prevented photooxidation, so that only a partial inhibition of photosynthetic oxygen evolution was found under these conditions.