In situ real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe Deinococcus radiodurans.

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.

Unusual radioresistance of nitrogen-fi xing cultures of Anabaena strains.

Nitrogen-fi xing cultures of two species of the fi lamentous, heterocystous cyanobacterium Anabaena, namely Anabaena sp. strain L-31 and Anabaena torulosa were found to be highly tolerant to 60Co gamma radiation. No adverse effect on diazotrophic growth and metabolism were observed up to a dose of 5 kGy. At higher doses, radiation tolerance showed a correspondence with the inherent osmotolerance, with Anabaena L-31 being the more radiation tolerant as well as osmotolerant strain. In Anabaena L-31, exposure to 6 kGy of gamma rays resulted in genome disintegration, but did not reduce viability. Irradiation delayed heterocyst differentiation and nitrogen fi xation, and marginally affected diazotrophic growth. All the affected parameters recovered after a short lag, without any discernible postirradiation phenotype. The radiation tolerance of these Gram-negative photoautodiazotrophs is comparable with that of the adiazotrophic photoautotrophic cyanobacterium Chroococcidiopsis or adiazotrophic heterotroph Deinococcus radiodurans. This is the fi rst report of extreme radioresistance in nitrogen-fi xing Anabaena cultures.

The Kdp-ATPase system and its regulation.

K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E.coli, the KdpATPase (comprising of the KdpFABC complex), encoded by the kdpFABC operon, is an inducible high-affinity K+ transporter that is synthesised under conditions of severe K+ limitation or osmotic upshift. The E.coli kdp expression is transcriptionally regulated by the KdpD and KdpE proteins, which together constitute a typical bacterial two-component signal transduction system. The Kdp system is widely dispersed among the different classes of bacteria including the cyanobacteria. The ordering of the kdpA, kdpB and kdpC is relatively fixed but the kdpD/E genes show different arrangements in distantly related bacteria. Our studies have shown that the cyanobacterium Anabaena sp. strain L-31 possesses two kdp operons, kdp1 and kdp2, of which, the later is expressed under K+ deficiency and desiccation. Among the regulatory genes,the kdpD ORF of Anabaena L-31 is truncated when compared to the kdpD of other bacteria, while a kdpE -like gene is absent. The extremely radio-resistant bacterium, Deinococcus radiodurans strain R1, also shows the presence of a naturally short kdpD ORF similar to Anabaena in its kdp operon. The review elaborates the expression of bacterial kdp operons in response to various environmental stress conditions, with special emphasis on Anabaena. The possible mechanism(s)of regulation of the unique kdp operons from Anabaena and Deinococcus are also discussed.

A novel potassium deficiency-induced stimulon in Anabaena torulosa.

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacterium Anabaena torulosa and of nine proteins in Escherichia 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

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 of Anabaena 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 Κ+, 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 N 2 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.

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 effi ciently 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 Ana baena 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.