A novel potassium deficiency-induced stimulon in Anabaena torulosa

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacteriumAnabaena torulosa and of nine proteins inEscherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K+ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase of A. torulosa and the glycine betaine-binding protein of E. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria

Nitrogen status and heat-stress-dependent differential expression of the cpn60 chaperonin gene influences thermotolerance in the cyanobacterium Anabaena

Heat stress caused rapid and severe inhibition of photosynthesis and nitrate reduction in nitrate-supplemented cultures of the cyanobacterium Anabaena sp. strain L-31, compared to nitrogen-fixing cultures. Anabaena strains harbour two hsp60 family genes, groEL and cpn60, respectively encoding the 59 kDa GroEL and 61 kDa Cpn60 chaperonin proteins. Of these two Hsp60 chaperonins, GroEL was strongly induced during heat stress, irrespective of the nitrogen status of the cultures, but Cpn60 was rapidly repressed and degraded in heat-stressed nitrate or ammonium-supplemented cultures. The recovery of photosynthesis, nitrate assimilation and growth in heat-stressed, nitrate-supplemented cultures were preceded by resynthesis and restoration of cellular Cpn60 levels. Glutamine synthetase activity, although adversely affected by prolonged heat stress, was not dependent on either the nitrogen status or Cpn60 levels during heat stress. Overexpression of the Cpn60 protein in the closely related Anabaena sp. strain PCC7120 conferred significant protection from heat stress to growth, photosynthesis and nitrate reduction in the recombinant strain. The data favour a role for Cpn60 in carbon and nitrogen assimilation in Anabaena

Sodium transport in filamentous nitrogen fixing cyanobacteria

Two filamentous, nitrogen fixing cyanobacteria were examined for their salt tolerance and sodium (Na+) transport. Anabaena torulosa, a saline form, grew efficiently and fixed nitrogen even at 150 mM salt (NaCl) concentration while, Anabaena L-31, a fresh water cyanobacterium, failed to grow beyond 35 mM NaCl. Anabaena torulosa showed a rapidly saturating kinetics of Na+ transport with a high affinity for Na+ (Km, 0.3 mM). Anabaena L-31 had a much lower affinity for Na+ (Km, 2.8 mM) than Anabaena torulosa and the pattern of uptake was somewhat different. Both Anabaena spp. exhibited an active Na+ extrusion which seems to be mediated by a Na+-K+ ATPase and aided by oxidative phosphorylation. Anabaena L-31 was found to retain much more intracellular Na+ than Anabaena torulosa. The results suggest that the saline form tolerates high Na+ concentrations by curtailing its influx and also by an efficient Na+ extrusion, although these alone may not entirely account for its success in saline environment

A model for cell type-specific differential gene expression during heterocyst development and the constitution of aerobic nitrogen fixation ability in Anabaena sp. strain PCC 7120

When deprived of combined nitrogen, aerobically-grown filaments ofAnabaena sp. strain PCC7120 differentiate specialized cells called the heterocysts. The differentiation process is an elaborate and well orchestrated programme involving sensing of environmental and developmental signals, commitment of cells to development, gene rearrangements, intricate DNA-protein interactions, and differential expression of several genes. It culminates in a physiological division of labour between heterocysts, which become the sole sites of aerobic nitrogen fixation, and vegetative cells, that provide photosynthate to the heterocysts in return for nitrogen supplies. We propose a model, to describe the chronology of the important events and to explain how cell type-specific differential gene expression is facilitated by DNA-protein interactions leading to the development of heterocysts and constitution of nitrogen-fixing apparatus in Anabaena

Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

Sodium 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 in Anabaena torulosa, Anabaena L-31 and Plectonema boryanum. The features of this requirement have been mainly studied in Anabaena 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 in Anabaena torulosa and Anabaena 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 in Anacystis 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

Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins

AbstractThe radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the ‘interstitial’ layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug

Nitrogen fixation genes (nif K,D,H) in the filamentous nonheterocystous cyanobacterium Plectonema boryanum do not rearrange

The organisation of the structural genes for nitrogen fixation (nif K,D and H) in a nonheterocystous, filamentous cyanobacterium Plectonema boryanum has been examined in comparison with a heterocystous cyanobacterium, Anabaena torulosa. DNA from repressed (fix-) cultures of A. torulosa showed a discontinuous nif region spread over approximately 18 kb, an arrangement typical of the vegetative cells of heterocystous cyanobacteria. The region contained a contiguousnif DH separated fromnif K. by nearly 11 kb DNA. The intervening 11 kb DNA harboured the genexis A involved in the rearrangement ofnif K,D,H to form a cluster during differentiation of heterocysts. DNA from Plectonema boryanum had a small, contiguous nif KDH cluster spanning a region of approximately 4 kb. DNA homologous to the 11 kb excison with its residentxis A was not present. Nif hybridisation patterns of restriction digests of the DNA isolated from repressed (fix-) or induced (fix-) cultures ofP. boryanum were completely identical. These results unequivocally demonstrate that in the nonheterocystous cyanobacterium, unlike in the heterocystous strains, no gene rearrangement, either within the nif KDII cluster or in its vicinity, accompanies the expression of nitrogenase activity

Engineering of Deinococcus radiodurans R1 for bioprecipitation of uranium from dilute nuclear waste

Genetic engineering of radiation-resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. We have constructed a Deinococcus radiodurans strain harboring phoN, a gene encoding a nonspecific acid phosphatase, obtained from a local isolate of Salmonella enterica serovar Typhi. The recombinant strain expressed an ~27-kDa active PhoN protein and efficiently precipitated over 90% of the uranium from a 0.8 mM uranyl nitrate solution in 6 h. The engineered strain retained uranium bioprecipitation ability even after exposure to 6 kGy of 60Co gamma rays. The PhoN-expressing D. radiodurans offers an effective and eco-friendly in situ approach to biorecovery of uranium from dilute nuclear waste

Overexpression of the groESL Operon Enhances the Heat and Salinity Stress Tolerance of the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC7120▿

The bicistronic groESL operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the Anabaena sp. strain PCC7120 genome. In the recombinant Anabaena strain, the additional groESL operon was expressed from a strong cyanobacterial PpsbA1 promoter without hampering the stress-responsive expression of the native groESL operon. The net expression of the two groESL operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant Anabaena strain to heat and salinity stresses

Possible amelioration of coastal soil salinity using halotolerant nitrogen-fixing cyanobacteria

A brackish-water, nitrogen-fixing cyanobacterium, Anabaena torulosa, could successfully grow and fix nitrogen on moderately saline "Kharland" soils (soil conductivity 5 to 8.50 dS m<SUP>-1</SUP>), typical of Indian coastline. During five weeks of growth under laboratory as well as field conditions, the cyanobacterium exhibited high rates of nitrogen fixation and substantially enriched the nitrogen status of saline soils (43-76%), although the fixed nitrogen remained confined to the cyanobacterial biomass. Most (&gt;90%) of the cell-bound Na<SUP>+</SUP> remained extracellularly trapped in the mucopolysaccharide sheath of A. torulosa; traces of the cation that permeated cyanobacterial cells were found to exist in an osmotically active, free state. No evidence was found for the incorporation of Na<SUP>+</SUP> into any biomolecule, especialty proteins or carbohydrates. Therefore, permanent removal of Na<SUP>+</SUP> from saline soils using cyanobacteria may not be possible, since Na<SUP>+</SUP> is released back into the soil subsequent to the death and decay of cyanobacteria. Removal of top soil containing cyanobacterial mats significantly decreased the soil salinity (between 26-38%). But such a practice removes all the fixed nitrogen and carbon and also does not seem feasible on a large scale. Amelioration of soil salinity by simultaneous application of A. torulosa during crop growth seems to be an attractive possibility, especially since it can also supplement the nitrogen requirement of the crop