The nitrate assimilation pathway and its regulation in the high-protein neutraceutical cyanobacterium, Arthrospira (Spirulina), were studied. A complete characterization of the genes of the nitrate uptake and assimilatory pathway in Arthrospira platensis strain PCC 7345 was done including cloning, sequencing, phylogenetic analysis and expression studies. Genomic localization studies revealed that their clustering is different from the operons known in other cyanobacteria; only nrtP and narB are organized together, while nirA, glnA and gltS exist in separate genomic locations. The presence of both types of nitrate transporters (nrtP/ABC types) in A. platensis is rare, as their occurrence is usually specific to marine and freshwater microorganisms, respectively. The positive effect of nitrate on transcript accumulation of narB, nirA and nrtP genes in N-depleted and N-restored cultures confirmed nitrate induction, which is abolished by the addition of ammonium ions into the medium. Gene expression studies in response to nitrate, nitrite, ammonium and glutamine provided the first evidence of differential regulation of multiple genes of nitrate assimilatory pathway in Arthrospira.
Unraveling the structure of complex biological networks and relating it to their functional role is an important task in systems biology. Here we attempt to characterize the functional organization of the large-scale metabolic networks of three microorganisms. We apply flux balance analysis to study the optimal growth states of these organisms in different environments. By investigating the differential usage of reactions across flux patterns for different environments, we observe a striking bimodal distribution in the activity of reactions. Motivated by this, we propose a simple algorithm to decompose the metabolic network into three subnetworks. It turns out that our reaction classifier, which is blind to the biochemical role of pathways, leads to three functionally relevant subnetworks that correspond to input, output, and intermediate parts of the metabolic network with distinct structural characteristics. Our decomposition method unveils a functional bow-tie organization of metabolic networks that is different from the bow-tie structure determined by graph-theoretic methods that do not incorporate functionality.
In order to assess the potential of Spirulina (Arthospira) platensis as a source of abundant, thermostable nitrate assimilatory enzymes, the specific activities and thermal tolerance of nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) were compared with those of rice in crude extracts in vitro. The results show that Spirulina enzymes have relatively higher thermotolerance. When the extracts were pre-exposed to 80 °C for 1 hr, Spirulina enzymes retained higher activities by 3.4, 1.7 and 3.7 fold, respectively than corresponding enzymes in rice. This property was not due to salts and other small proteins/molecules, as their removal by gel filtration (G-25) did not affect their thermotolerance.
Nitrate response at the plant level is mediated by the transcriptional regulation of several hundreds of genes, but no common cis-acting nitrate-responsive elements (NREs) have been identified so far. Earlier, we bioinformatically ruled out the possibility that the previously published [(a/t)7Ag/cTCA] motif could act as NRE on its own (Das et al., 2007, Mol. Genet. Genomics, 278: 519-525). In the present study, we examined other motifs such as Dof and GATA binding elements in homologous as well as heterologous pairwise combinations in the Arabidopsis genome in silico. None of the above three motifs revealed any unique association with nitrate responsive genes or their subsets in any combination, either within their ORFs or 1 kb flanking sequences on either side. Additionally, twelve new, top-scoring candidate motifs that were generated using different online motif samplers were analyzed in silico using a subset of 21 early nitrate responsive genes, but did not reveal any specificity of occurence. These results underscore the need to continue the search for novel candidate NREs, as possible sites of intervention to understand/improve nitrate-responsive gene expression and nitrate use efficiency.
[This corrects the article DOI: 10.1007/s12298-008-0017-z.].
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