Protein fingerprinting may serve as a complementary tool for the phylogenetic classification of heterocystous (Nostoc, Anabaena, Cylindrospermum, Aulosira and Tolypothrix) Cyanobacteria

摘要

A combination of morphology, SDS-PAGE, 16S rRNA gene and the fuzzy approach has been used for the first time to attest the phylogenetic affiliation of the cyanobacterial species such as Nostoc, Anabaena, Cylindrospermum, Tolypothrix and Aulosira. The idea was to ascertain as how far SDS-PAGE analysis and morphological taxonomy work coherently and serve as a reliable tool, compatible with 16S rRNA gene based classification of cyanobacteria. Intermingling of Nostoc and Anabaena species in SDS-PAGE and 16S rRNA gene indicates that the two taxa are not clearly separated at the genetic level and may be polyphyletic in origin. In contrast to this, clustering of Tolypothrix and Aulosira species in the same clade attests the taxonomic coherence of these species. However, Cylindrospermum showed greater phylogenetic relatedness with Anabaena strain in all phylogenetic analyses. Despite a few minor incongruities, protein fingerprint depicted greater similarity with 16S rRNA gene as compared to morphological taxonomy. Thus protein fingerprinting developed using fuzzy approach appears to be more compatible with 16S rRNA gene analysis than morphological taxonomy in discriminating cyanobacteria up to the species level. Introduction Cyanobacteria are the most widespread, photosynthetic prokaryotes with remarkable degrees of morphological and developmental diversity. Among the heterocystous cyanobacteria, Nostocalean strains are considered to be the most important component of the N2-fixing community in nutrient poor, arid and semiarid soils world wide (Doods et al., 1995; Potts, 2000; Wynn-Williams, 2000; Bhatnagar and Bhatnagar, 2005). Cyanobacteria have been traditionally classified on the basis of their morphological and physiological characteristics (Gietler, 1932; Desikachary, 1959). However, morphology of the strains may change depending on environmental conditions and the diversity of the strains can be altered by selective culture conditions (Palinska et al., 1996). For example the strains of Nodularia were found to loose gas vesicles (Lehtim?ki et al., 2000) and Aphanizomenon flos-aquae formed colony (Gugger et al., 2002) in their culture conditions. Komárek & Anagnostidis (1989) have estimated that more than 50% of the strains in culture collections are misidentified. To overcome these limitations, molecular method based on the use of small subunit rRNA gene has been proposed (Woese, 1987). The conservative nature of 16S rRNA gene, its universal distribution and the vast availability of sequence information in public databases (Genbank, EMBL, DDBJ and RDP) make it the marker of choice for taxonomical studies (Weisburg et al., 1991). The classification based on 16S rRNA gene analysis has been often compared with morphological identification and suffers from several limitations. Palinska et al., (1996) demonstrated that the divergence of the genera Synechococcus, Synechocystis, Merismopedia and Eucapsis based on morphological characters was not reflected in genetic diversity. Thus there is a need to develop another marker, which can complement the 16S rRNA gene based phylogeny in a more reliable manner as compared to morphology.SDS-PAGE whole cell protein pattern analysis has emerged as a powerful tool for bacterial identification having the advantages of being fairly fast and easy and, when performed under highly standardized conditions, it offers a better taxonomic resolution at species or subspecies level (Vandamme et al., 1996; Derbyshire and Whitton, 1968; Kersters and De Ley, 1975; Elliot et al., 1993). The SDS-PAGE protein profiles analysis has been successfully used to characterize various species of Lactobacillus and Leuconostoc (Perez et al., 2000), Staphylococcus (Berber et al., 2003) and thermophillic bacteria (Liu et al., 2006). However, the use of this technique in the characterization of cyanobacteria is still debatable. Palinska et al., (1996) used it for the first time to characterize Merismopedia and suggested it to