Genetic diversity and classification of cyanobacteria in different Azolla species by the use of PCR fingerprinting. Theor Appl Genet

Stockholm University, Tukholma, Stockholm, Sweden
Theoretical and Applied Genetics (Impact Factor: 3.79). 10/1999; 99(7):1187-1193. DOI: 10.1007/s001220051323


Symbiotically associated cyanobacteria from 18 accessions within all known species in the genus Azolla were examined and classified by the use of polymerase chain reaction (PCR)-fingerprinting. A repetitive sequence specific
for cyanobacteria, the short tandemly repeated repetitive (STRR) sequence, was used as a primer in the reaction. Cyanobacterial
filaments isolated directly from the Azolla leaf cavity or contained within homogenised symbiotic Azolla tissue were used as templates. Based on the fingerprint pattern, distinct differences were demonstrated between cyanobacteria
isolated from the Euazolla and Rhizosperma sections. In addition, individual fingerprints were obtained from all cyanobacteria isolated from the different Azolla species. The fingerprints were used to generate a phylogenetic tree. Three clusters were distinguished: one contained the
four isolates from the section Euazolla, a second the isolate from Azolla filiculoides, and a third the three isolates from the section Rhizosperma. By the use of STRR-PCR fingerprinting, new data on the taxonomy of cyanobacteria in Azolla were obtained, which have been difficult to generate by other classification methods. PCR-fingerprinting may, therefore,
be a valuable tool for diversity and classification studies of symbiotic cyanobateria from Azolla and, as co-evolution between the cyanobacteria and its corresponding host exists the method may also be useful for the taxonomy
of Azolla.

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    • "Others have suggested that in addition to a major nitrogen-fixing, noncultivable cyanobiont, Azolla harbors one or more cultivable minor symbiotic cyanobacteria (Meeks et al. 1988, Gebhardt and Nierzwicki-Bauer 1991, Kim et al. 1997). Many studies have also analyzed the diversity among the cyanobionts from diverse Azolla species, varieties, or ecotypes, using immunological techniques (Ladha and Watanabe 1982, Liu et al. 1986, 1989), lectin hemagglutination techniques (McCowen et al. 1987), DNA hybridization methods, DNA fingerprints (Nierzwicki-Bauer and Haselkorn 1986, Meeks et al. 1988, Plazinski et al. 1990, Van Coppenolle et al. 1995, Zheng et al. 1999), and fatty acid comparison (Caudales et al. 1995). Our aim in this study was to investigate the diversity and host specificity of cyanobionts from individual Azolla species and strains by determining whether the same cyanobacterial genotype was always present in a particular species of Azolla, regardless of geographic origin. "
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    ABSTRACT: A unique, hereditary symbiosis exists between the water fern Azolla and cyanobacteria that reside within a cavity in the dorsal leaf-lobe of the plant. This association has been studied extensively, and questions have frequently been raised regarding the number and diversity of cyanobionts (cyanobacterial symbionts) among the different Azolla strains and species. In this work, denaturating gradient gel electrophoresis (DGGE) and a clone library based on the 16S rRNA gene were used to study the genetic diversity and host specificity of the cyanobionts in 35 Azolla strains covering a wide taxonomic and geographic range. DNA was extracted directly from the cyanobacterial packets, isolated after enzymatic digestion of the Azolla leaves. Our results indicated the existence of different cyanobiont strains among Azolla species, and diversity within a single Azolla species, independent of the geographic origin of the host. Furthermore, the cyanobiont exhibited host-species specificity and showed most divergence between the two sections of genus Azolla, Azolla and Rhizosperma. These findings are in agreement with the recent redefinition of the taxon Azolla cristata within the section Azolla. With regard to the taxonomic status of the cyanobiont, the genus Anabaena of the Nostocaceae family was identified as the closest relative by this work.
    Full-text · Article · Feb 2008 · Journal of Phycology
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    • "Hence, in the context of the endosymbiotic theory, according to which chloroplasts evolved from an ancient monophyletic symbiosis between a cyanobacterium and a pigment-free eukaryote, the cyanobiont of Azolla may be evolving towards becoming a nitrogen-fixing 'organelle'. This is also supported by PCR-based DNA fingerprinting which shows that the association is highly specific and that each Azolla species associates with one specific cyanobacterial strain irrespective of geographical origin (Zheng et al., 1999). "
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    ABSTRACT: Cyanobacteria are able to form stable nitrogen-fixing symbioses with diverse eukaryotes. To extend our understanding of adaptations imposed by plant hosts, two-dimensional gel electrophoresis and mass spectrometry (MS) were used for comparative protein expression profiling of a cyanobacterium (cyanobiont) dwelling in leaf cavities of the water-fern Azolla filiculoides. Homology-based protein identification using peptide mass fingerprinting [matrix-assisted laser desorption ionization-time of flight (MALDI-TOF-MS)], tandem MS analyses, and sequence homology searches resulted in an identification success rate of 79% of proteins analysed in the unsequenced cyanobiont. Compared with a free-living strain, processes related to energy production, nitrogen and carbon metabolism, and stress-related functions were up-regulated in the cyanobiont while photosynthesis and metabolic turnover rates were down-regulated, stressing a slow heterotrophic mode of growth, as well as high heterocyst frequencies and nitrogen-fixing capacities. The first molecular data set on the nature of the NifH post-translational modification in cyanobacteria was also obtained: peptide mass spectra of the protein demonstrated the presence of a 300-400 Da protein modification localized to a specific 13 amino acid sequence, within the part of the protein that is ADP-ribosylated in other bacteria and close to the active site of nitrogenase. Furthermore, the distribution of the highest scoring database hits for the identified proteins points to the possibility of using proteomic data in taxonomy.
    Preview · Article · Feb 2008 · Journal of Experimental Botany
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    • "The eight cyanobacteria were grown in BG11 0 medium (Stainer et al. 1971) at 28°C under continuous shaking and an illumination at 18 lmol photons m À2 s À1 . A. filiculoides was grown in a pond in the greenhouse and the cyanobacteria were isolated after surface sterilization of the leaves by carefully removing the cyanobacteria from the leaf cavity with a needle, and then kept at À20°C until used directly in the PCR reactions as described by Zheng et al. (2002). "
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    ABSTRACT: A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.
    Full-text · Article · Feb 2005 · Archives of Microbiology
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