ABSTRACT
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.
Subject(s)
Cyanobacteria/genetics , Cyanobacteria/metabolism , Gene Expression Regulation, Bacterial , Nitrates/metabolism , Ammonium Compounds/pharmacology , Anion Transport Proteins/genetics , Cyanobacteria/classification , Enzyme Activation/drug effects , Gene Expression Regulation, Bacterial/drug effects , Gene Order , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/metabolism , Glutamine/pharmacology , Molecular Sequence Data , Nitrate Reductase/genetics , Nitrate Reductase/metabolism , Nitrate Transporters , Nitrates/pharmacology , Nitrite Reductases/genetics , Nitrite Reductases/metabolism , Nitrites/metabolism , Nitrites/pharmacology , PhylogenyABSTRACT
Arthrospira (Spirulina) platensis is widely used as a food supplement and has been an economically important species for centuries. However, the genetic aspect of studies of this particular organism has always been neglected, mainly because of the nonavailability of suitable methods for isolation of nucleic acids and the difficulties faced during further manipulations. Although total RNA has been isolated using commercially available kits, we present a method optimized to obtain DNA-free total RNA of higher yields and higher purity in less time than is required by other methods (<2 h). It involves hot phenol - chloroform - IAA extraction using an aqueous to organic phase ratio of 1:2 followed by lithium chloride precipitation and 70% ethanol wash. This method, optimized for the cyanobacterium Arthrospira (Spirulina) platensis, eliminates the need for DNase treatment and produces high-quality RNA, as validated by bioanalyzer, RT-PCR, and cloning. With the recent release of the Arthrospira genome, the current method will be of great value for carrying out high-throughput studies like microarray and real-time PCR.
Subject(s)
Cyanobacteria/genetics , Genetic Techniques , RNA, Bacterial/isolation & purification , Polymerase Chain Reaction , RNA, Bacterial/geneticsABSTRACT
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.
