Molecular analysis of genes involved in carbohydrate metabolism in the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584

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Virginia Tech

Synthesis of water stress proteins (Wsp) is induced upon repeated desiccation and rehydration of immobilized cells of the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584 (Nostoc 584). The Wsp polypeptides synthesized and secreted by field material of N. commune have been extensively characterized and shown to exist as three isoforms with molecular masses of 33, 37 and 39 kDa. In order to understand the role of Wsp in the mechanism of tolerance to water stress an attempt was made to isolate the gene(s) that encodes Wsp in Nostoc 584. A polyclonal antibody raised against a mixture of the isoforms was used to screen expression libraries (phage and plasmid) of Nostoc 584 genomic DNA fragments. This work presents the analysis of clones, isolated from the expression libraries, which cross react with Wsp antiserum. Sequencing of the DNA from one of the cross reacting clones revealed an incomplete open reading frame (ORFA) that showed strong similarity to two fructose bisphosphate aldolases, CfxA and CfxB, from the photosynthetic purple non-sulfur bacterium, Rhodobacter sphaeroides. A promoter region present upstream of ORFA is recognized by RNA polymerase from E. coli. Further upstream of the promoter lies trnK encoding lysyl-tRNA, identified by evident similarity to the corresponding gene from the chloroplast of the liverwort, Marchantia polymorpha. The remainder of the aldolase gene (fba) was isolated using the colony hybridization technique. Sequence analysis of DNA from the second cross reactive clone revealed six ORFs (ORFs 1 through 6).The products of ORFI and ORF2 are overproduced in this clone. The polypeptide encoded by ORFI shows very strong cross-reactivity with the polyclonal Wsp antibody, whereas ORF2 does not. Database searches using the deduced amino acid sequences of the six ORFs have provided clues to the possible identities of these ORFs. ORF6 shows correspondence with a protein, in Arabidopsis thaliana, which is induced in response to cold, abscissic acid and water stress. The common feature shared by ORFs 1 to 5 is that the highest similarities are observed with enzymes involved in carbohydrate metabolism. ORFs 1 through 5 may possibly represent a novel cluster of genes that form all or part of an operon involved in the metabolism of carbohydrates in Nostoc 584. Fructose bisphosphate aldolase (EC is a key enzyme in carbohydrate metabolism, playing a role in glycolysis as well in the Calvin cycle of carbon dioxide fixation. The potential roles played by aldolase and the products of ORFs 1 through 5 in overall carbon metabolism of Nostoc commune UTEX 584 are discussed.

water stress proteins