Joseph Thomas

Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

AbstractSodium affects the metabolism of eukaryotes and prokaryotes in several ways. This review collates information on the effects of Na+ on the metabolism of cyanobacteria with emphasis on the N2,fixing filamentous species. Na+ is required for nitrogenase activity inAnabaena torulosa, Anabaena L-31 andPlectonema boryanum. The features of this requirement have been mainly studied inAnabaena torulosa. The need for Na+ is specific and cannot be replaced by K+, Li+, Ca 2 + or Mg2+. Processes crucial for expression of nitrogenase such as molybdenum uptake, protection of the enzyme from oxygen inactivation and conformational activation of the enzyme are not affected by Na+. Mo-Fe protein and Fe protein, the two components of nitrogenase are synthesized in the absence of Na+ but the enzyme complex is catalytically inactive. Photoevolution of O2 and CO2 fixation, which are severely inhibited in the absence of Na+, are quickly restored by glutamine or glutamate indicating that Na+ deprivation affects photosynthesis indirectly due to deficiency in the products of N2 fixation. Na+ deprivation decreases phosphate uptake, nucleoside phosphate pool and nitrogenase activity. These effects are reversed by the addition of Na+ suggesting that a limitation of available ATP caused by reduced phosphate uptake results in loss of nitrogenase activity during Na+ starvation.Na+ influx inAnabaena torulosa andAnabaena L-31 is unaffected by low K+ concentration, is carrier mediated, follows Michaelis-Menten kinetics and is modulated mainly by membrane potential. Treatments which cause membrane depolarisation and hyperpolarisation inhibit and enhance Na+ influx respectively. These cyanobacteria exhibit rapid active efflux of Na+, in a manner different from the Na+/H+ antiporter mechanism found inAnacystis nidulans. Na+ requirement in nitrogen metabolism including nitrate assimilation, synthesis of amino acids and proteins, in respiration and oxidative phosphorylation, in transport of sugars and amino acids, cellular distribution of absorbed sodium, physiological basis of salt tolerance and prospects of reclamation of saline soils by cyanobacteria are the other aspects discussed in this review.

The initial organic products of fixation of 13N-labeled nitrogen gas by the blue-green alga Anabaena cylindrica

Methods have been developed for the rapid isolation and characterization of the first organic products of fixation of 13N-labeled N2. In experiments with the blue-green alga, Anabaena cylindrica, glutamine is the first 13N-labeled organic product observed, and glutamate is the second. The results indicate that the glutamine synthetase/glutamate synthase pathway is operative in this blue-green alga.

Sodium is required for nitrogenase activity in cyanobacteria

The cyanobacteriaAnabaena torulosa andAnabaena L-31 require sodium (Na+) for growth on N2 but not in the presence of NH4+. Na+-starved cultures show relatively little or no nitrogenase activity although they differentiate normal heterocysts. Nitrogenase activity appears rapidly on addition of Na+ to Na+-starved cultures. The time course of appearance of activity after addition of Na+ suggests that Na+ is involved in activation of the existing enzyme rather than in its de novo synthesis.

Effect of sodium on nitrogen fixation in Anabaena torulosa and Plectonema boryanum

The cyanobacterium Anabaena torulosa required sodium and molybdenum for nitrogenase activity and diazotrophic growth. Addition of sodium or molybdenum to cultures deficient in either element restored nitrogenase activity. Heterocyst differentiation was unaffected by sodium but molybdenum deficiency enhanced differentiation. The non-heterocystous cyanobacterium Plectonema boryanum 594 also required sodium for nitrogenase activity but synthesized presumptive nitrogenase component I and II proteins during sodium deficiency. The results show that in cyanobacteria nitrogenase is synthesized even in the absence of sodium but functions only in its presence.

Dual Binding Sites for Peanut Lectin on Rhizobia

Summary: Purified peanut lectin (peanut agglutinin, PNA) labelled with fluorescein isothiocyanate (FITC-PNA) or iodine-125 (125I-PNA) bound to Rhizobium B. TG-3 and Rhizobium 5a, which nodulate peanut, but did not bind to R. japonicum or R. meliloti, which do not nodulate peanut, or to the non-nitrogen-fixing bacteria Escherichia coli and Bacillus subtilis. Washing the rhizobia in phosphate-buffered saline markedly decreased PNA binding. The decrease in binding could be correlated with removal of exopolysaccharides during washing. Dialysed culture filtrate could be labelled with 125I-PNA and the radioactivity was recovered in an acetone-precipitable exopolysaccharide fraction, showing that the lectin complexed with exopolysaccharides. Residual binding which remained after washing could not be removed by further washing. Moreover, rhizobia could be labelled with 125I-PNA after removal of exopolysaccharides and the radioactivity was recovered by extraction with hot phenol, indicating that the lectin complexed also with lipopolysaccharide on the bacterial cell wall. Rhizobial lipopolysaccharide not only inhibited haemagglutination by PNA but also dispersed preagglutinated human erythrocytes into free cell suspensions. The results demonstrate the presence of dual binding sites for PNA on rhizobia. Exopolysaccharides are the major sites and the remainder are lipopolysaccharides.

Legume-Rhizobium interactions: role of cowpea root exudate in polysaccharide synthesis and infectivity of Rhizobium species

Nodulation pattern of cowpea (Vigna sinensis) was markedly influenced by the growth phase of the microsymbiont, Rhizobium sp. strain 1001. Rhizobia from stationary growth phase cultures required much longer time after inoculation to initiate successful infections on cowpea seedlings than the rhizobia from log phase. Preincubation of stationary phase rhizobia in root exudate of cowpea seedlings grown without NH4+in the medium enhanced the formation of capsules around a large proportion of the cells. The longer the time of incubation the larger was the proportion of cells with capsules. The increase in cell-surface polysaccharide content showed, up to a limit, a positive relationship with the increase in percentage of plants scored for faster nodulation response. Root exudate of cowpea plants grown with NH4+could neither stimulate polysaccharide synthesis by the rhizobia from stationary phase nor promote them to initiate infections without delay, indicating the regulatory role of combined nitrogen in early infection events. Root exudates of Vigna radiata (mung bean) grown without NH4+also promoted polysaccharide synthesis by Rhizobium sp. strain 1001 and faster nodulation on cowpea plants, but the exudates of Trigonella foenum-graecum and Medicago sativa were not effective indicating the relative specificity of the early interactions.

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31☆

Abstract In cell-free preparations of NH 4 + -grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N 2 -grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH 4 + -mediated repression. Glutamine synthetase activity in both N 2 -grown and NH 4 + -grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N 2 -grown cultures, a drastic decrease in the enzyme activity by exogenous NH 4 + can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K m values for NH 4 + , glutamate Mg 2+ , and ATP similar to those observed for the enzyme from N 2 - and NH 4 + -grown Anabaena , but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH 4 + -mediated modification to a less active form may be a means of regulation of glutamine synthetase in N 2 -fixing cultures of Anabaena .

Extracellular polypeptides ofAnabaena L-31: Evidence for their role in regulation of heterocyst formation

Extracellular polypeptides released by both N2-grown [peptide I] and NO3-grown [peptide II]Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction yielded only 3 amino acids. On addition to a freshly inoculated N2-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be relieved by methionine sulphoximine, which by itself enhances differentiation, but was greatly relieved by addition of the peptide I fraction. The data suggest but does not prove, thatAnabaena L-31 synthesises “inducer” or “inhibitor” peptides which could possibly control pattern formation.

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.

MICROSPECTROPHOTOMETRIC STUDIES ON THE PIGMENTS IN VIVO OF SINGLE ALGAL CELLS‐I. PIGMENTS OF CHLORELLA PYRENOIDOSA

Abstract— The pigments in vivo of single cells of Chlorella pyrenoidosa were studied by the microspectrophotometric technique. An accessory recording the first derivative of absorption was used to obtain fine resolution and enhanced accuracy. The results suggest that there are several long‐wavelength components of Chl a in vivo. In addition, there seem to be four short‐wave forms of Chl a. It is also likely that Chl b exists in vivo in two different forms. The existence of all these forms was demonstrated at room temperature.