Cyanobacterium Nostoc commune can tolerate the simultaneous stresses of desiccation, UV irradiation, and oxidation. Acidic WspA, of ϳ33.6 kDa, is secreted to the three-dimensional extracellular matrix and accounts for greater than 70% of the total soluble protein. The wspA gene of N. commune strain DRH1 was cloned and found in a single genomic copy, in a monocistronic operon. Transcription of wspA and sodF (superoxide dismutase), and synthesis and secretion of WspA, were induced upon desiccation or UV-A/B irradiation of cells. Recombinant WspA binds the UV-A/B absorbing pigment scytonemin through non-covalent interactions. WspA peptide polymorphism, and heterogeneity of multiple wspA sequences within cells of a single colony, account for distinct WspA isoforms. WspA has no similarity to entries in the sequence databases and wspA, a possible xenolog, is restricted to a subset of strains in the "form species" N. commune characterized through group I intron phylogeny. We hypothesize that WspA plays a central role in the global stress response of N. commune through modulation of the structure and function of the three-dimensional extracellular matrix, particularly the transport, distribution, and/or macromolecular architecture of mycosporine and scytonemin UV-A/B absorbing pigment complexes.Cyanobacteria make a major contribution to world photosynthesis and nitrogen fixation, but their dense and often toxic "blooms" in marine, brackish, and fresh waters are of growing concern to public health (1). The sequences of a wide range of cyanobacterial genomes are becoming available and constitute an important resource both for the rational management and exploitation of these organisms, as well as the study of topics as diverse as carbon fixation, cell differentiation, plant and fungal symbioses, secondary product biosynthesis, and evolution.

No abstract

Genomic DNA of Nostoc commune (Cyanobacteria) became covalently modified during decades of desiccation. Amplification of gene loci from desiccated cells required pretreatment of DNA with N-phenacylthiazolium bromide, a reagent that cleaves DNA- and protein-linked advanced glycosylation end-products. DNA from 13 year desiccated cells did not show any higher levels of the commonly studied oxidatively modified DNA damage biomarkers 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxyuracil, compared to commercially available calf thymus DNA. Different patterns of amplification products were obtained with DNA from desiccated/rehydrating cells and a liquid culture derived from the dried material, using the same set of primers. In contrast, a reproducible fingerprint was obtained, irrespective of time of rehydration of the DNA, using a primer (5'-GWCWATCGCC-3') based upon a highly iterated palindromic repeat sequence present in the genome. In vitro, the desiccation of cccDNA led to loss of supercoiling, aggregation, loss of resolution during agarose gel electrophoresis and loss of transformation and transfection efficiency. These changes were minimized when DNA was desiccated and stored in the presence of trehalose, a non-reducing disaccharide present in Nostoc colonies. The response of the N.commune genome to desiccation is different from the response of the genomes of cyanobacteria and Deinococcus radiodurans to ionizing radiation.

The form species concept for the Cyanobacteria was evaluated using a comprehensive set of Nostoc samples that were collected during the past two centuries, from all continents, including regions from the Tropics to the Poles. Phylogenies were constructed based upon the conserved regions of tRNA Leu (UAA) group I intron DNA sequences. Thirty-four forms contained a tRNA Leu (UAA) intron of 284 nt. These 284-nt introns contained 200 nt of conserved sequence that, in most cases, shared 100 % sequence identity, they had three variable regions (I, II and III) amounting to 84 nt, contained no hypervariable region and formed a discrete cluster in phylogenetic analysis. These forms represented 31 independent populations in both hemispheres and constitute examples of form species Nostoc commune. Multiple introns were obtained from several of the populations. Ten populations contained introns of 287-340 nt with a hypervariable region, 8 to 59 nt in length, located between variable regions I and II. Alignments identified 15 examples where 5'-AAAAUCC-3' occurred at the hypervariable region-variable region II boundary ; this sequence is identical to the conserved sequence at the 3' intron-exon boundary (splice site) within the tRNA Leu (UAA) gene. The possibility that hypervariable regions were removed from the primary intron through secondary splicing was tested in vitro but proved to be negative under the experimental conditions used. Shared morphologies of genetically different strains, dissimilar morphologies in strains that share identical genetic markers, incorrect naming of culture collection strains and genetic drift in cultured strains emphasize that the successful delineation of cyanobacterial species requires the application of multiple taxonomic criteria.

Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (PAO stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.Loss of even a small fraction of intracellular water is lethal for most cells. Nevertheless, some cells, including many microbial pathogens of humans, survive extreme desiccation, often for protracted periods (1,22). The maximal longevity of microorganisms in a metabolically inactive, desiccated state is unknown (16,22,25), but there have been controversial (10, 21, 24) reports of ancient, yet viable bacteria in 25 million-to 40-million-year-old Dominican amber (2,17).Understanding the mechanisms that some organisms use to withstand the removal of virtually all of their water is an important problem in cell biology. The nonreducing disaccharides sucrose and trehalose protect membranes and proteins in vitro from dehydration damage, as described by the "water replacement hypothesis" (3). Survival of dehydration damage in a variety of organisms is correlated with intracellular accumulation of one of these disaccharides, and even the addition of exogenous trehalose or sucrose to cells that are sensitive to drying can increase survival (18,22).It has been proposed that the ability to survive desiccation may be conferred by transfection of desiccation-sensitive cells with genes which permit synthesis of trehalose or sucrose (4). This seems eminently practical since synthesis of either disaccharide requires only two steps, involves only two gene products, a synthase (reaction 1) and a phosphatase (reaction 2), and requires substrates found in all cells:Suc-6-P3Suc ϩ P I and Tre-6-P3Tre ϩ P IAn attempt to develop this approach in plants (13) was challenged because in this system survival of dehydration is not easily estimated and direct effects of a disaccharide on physical properties of dry tissue cannot be detected readily (9). In addition, trehalases are widespread in plants, which complicates the issue further (20). In the present study we used desiccation-sensitive Escherichia coli as a model to address these problems. MATERIALS AND METHODSBacterial strains, plasmids, and media. E. coli BL21DE3 (Invitrogen, Carlsbad, Calif.) has a chromosomal copy of the T7 RNA polymerase gene under control of the isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible lacUV5 promoter. Derivative strain BL21DE3(pT7-7) contains expression vector pT7-7, which has a T7 promoter that permits high-level gene expression after induction with IPTG. Strain BL21DE3(pSpsA) contains the sucrose-6-phosphate synthase (spsA) gene from the cyanobacterium Synechocystis sp. strain PCC 6803 cloned in pT7-7.Cloning of spsA. The following two primers we...