Molecular phylogeny and evogenomics of heterocystous cyanobacteria using rbcl gene sequence data

Taxonomic affiliations and molecular diversity of 41 heterocystous cyanobacteria representing 12 genera have been assessed on an evolutionary landscape using rbcl gene sequence data-based phylogenomics and evogenomics approaches. Phylogenetic affiliations have clearly demonstrated the polyphyly of the true branching cyanobacteria, along with a frequent intermixing amongst the heterocystous cyanobacteria. The monophyletic origin of the heterocystous cyanobacteria was also quite evident from maximum parsimony and neighbor joining analyses. Incongruency with the traditional scheme of cyanobacterial taxonomy was frequently observed, thus advocating towards some re-amendments in the cyanobacterial classificatory schemes. Evogenomics analyses of gene sequence data gave a clear indication about the greater evolutionary pace of the unbranched cyanobacteria as compared to the branched forms. It was evident that the order Nostocales would be controlling the future pace of evolution of heterocystous cyanobacteria. The cyanobacteria Nostoc was found to have the greatest genetic heterogeneity amongst the studied genera, along with some evidence towards events of lateral gene transfer amongst the heterocystous cyanobacteria in case of the rbcl gene. Thus, heterocystous cyanobacteria were found to be a fast evolving group, with estimates of gene conversion tracts pointing towards the unbranched heterocystous cyanobacteria being at the base of evolutionary diversifications of the complete heterocystous lineage.     

Nitrogen fixation genes (nif K,D,H) in the filamentous nonheterocystous cyanobacteriumPlectonema boryanum do not rearrange

The organisation of the structural genes for nitrogen fixation (nif K,D and H) in a nonheterocystous, filamentous cyanobacteriumPlectonema boryanum has been examined in comparison with a heterocystous cyanobacterium,Anabaena torulosa. DNA from repressed (fix-) cultures ofA. torulosa showed a discontinuousnif region spread over approximately 18 kb, an arrangement typical of the vegetative cells of heterocystous cyanobacteria. The region contained a contiguousnif DH separated fromnif K. by nearly 11 kb DNA. The intervening 11 kb DNA harboured the genexis A involved in the rearrangement ofnif K,D,H to form a cluster during differentiation of heterocysts. DNA fromPlectonema boryanum had a small, contiguousnif KDH cluster spanning a region of approximately 4 kb. DNA homologous to the 11 kb excison with its residentxis A was not present.Nif hybridisation patterns of restriction digests of the DNA isolated from repressed (fix^-) or induced (fix^--) cultures ofP. boryanum were completely identical. These results unequivocally demonstrate that in the nonheterocystous cyanobacterium, unlike in the heterocystous strains, no gene rearrangement, either within thenif KDII cluster or in its vicinity, accompanies the expression of nitrogenase activity.     

Phylogenetic comparison among the heterocystous cyanobacteria based on a polyphasic approach

Phylogenetic comparison has been done among the selected heterocystous cyanobacteria belonging to the sections IV and V. The hierarchical cluster analysis based on antibiotics sensitivity showed a distant relationship between the members of Nostocales and Stigonematales. Thus, multiple antibiotic resistance pattern used as marker provide easy, fast, and reliable method for strain discrimination and genetic variability. However, morphological, physiological (both based on principal component analysis) and biochemical analysis grouped true branching cyanobacteria along with the members of section IV. Molecular analysis based on 16S rRNA gene sequences revealed that Hapalosiphon welwitschii and Westiellopsis sp. were grouped in cluster I whereas Scytonema bohnerii, a false branching genera showed a close proximity with Calothrix brevissima in cluster II. Cluster III of clade 2 included Nostoc calcicola and Anabaena oryzae which proved the heterogeneity at the generic level. Cluster IV the largest group of clade 2 based on 16S rRNA gene sequences includes six strains of the genera Nostoc, Anabaena, and Cylindrospermum showing ambiguous evolutionary relationship. In cluster IV, Anabaena sp. and Anabaena doliolum were phylogenetically linked by sharing 99% sequence similarity. Probably, they were of the same genetic makeup but appear differently under the diverse physiological conditions. Section IV showed polyphyletic origin whereas section V showed monophyletic origin. Results suggested that either morphological or physiological or biochemical or molecular attribute is not sufficient to provide true diversity and phylogeny of the cyanobacteria at the generic level and thus, a polyphasic approach would be more appropriate and reliable.     

Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

Contiguous organization of nitrogenase genes in a heterocystous cyanobacterium

The organization of the three structural nitrogen fixation (nif) genes that encode nitrogenase (nif K and nif D) and nitrogenase reductase (nif H) have been examined in a number of cyanobacteria. Hybridization of Anabaena 7120 nif gene probes to restriction endonuclease-digested genomic DNA has shown (a) that cyanobacteria incapable of N₂ fixation have no regions of DNA with significant homology to the three nif probes, (b) that Pseudanabaena sp., a nonheterocystous cyanobacterium, has a contiguous nif KDH gene cluster, and (c) that in contrast with other heterocystous cyanobacteria, Fischerella sp. has a contiguous nif KDH gene cluster.     

Molecular identification and evolution of the cyclic peptide hepatotoxins, microcystin and nodularin, synthetase genes in three orders of cyanobacteria

The cyanobacterial hepatotoxins, microcystin and nodularin, are produced by a wide range of cyanobacteria. Microcystin production has been reported in the four cyanobacterial orders: Oscillatoriales, Chroococcales, Stigonematales, and Nostocales. The production of nodularin is a distinct characteristic of the Nostocales genus Nodularia. A single rapid method is needed to reliably detect cyanobacteria that are potentially capable of producing these hepatotoxins. To this end, a PCR was designed to detect all potential microcystin and nodularin-producing cyanobacteria from laboratory cultures as well as in harmful algal blooms. The aminotransferase (AMT) domain, which is located on the modules mcyE and ndaF of the microcystin and nodularin synthetase enzyme complexes, respectively, was chosen as the target sequence because of its essential function in the synthesis of all microcystins as well as nodularins. Using the described PCR, it was possible to amplify a 472 bp PCR product from the AMT domains of all tested hepatotoxic species and bloom samples. Sequence data provided further insight into the evolution of the microcystin and nodularin synthetases through bioinformatic analyses of the AMT in microcystin and nodularin synthetases, with congruence between the evolution of 16S rRNA and the AMT domain.     

THE CONTRIBUTION OF SUB‐SAHARAN AFRICAN STRAINS TO THE PHYLOGENY OF CYANOBACTERIA: FOCUSING ON THE NOSTOCACEAE (NOSTOCALES,CYANOBACTERIA)1

To date, phylogenies have been based on known gene sequences accessible at GenBank, and the absence of many cyanobacterial lineages from collections and sequence databases has hampered their classification. Investigating new biotopes to isolate more genera and species is one way to enrich strain collections and subsequently enhance gene sequence databases. A polyphasic approach is another way of improving our understanding of the details of cyanobacterial classification. In this work, we have studied phylogenetic relationships in strains isolated from freshwater bodies in Senegal and Burkina Faso to complement existing morphological and genetic databases. By comparing 16S rDNA sequences of African strains to those of other cyanobacteria lineages, we placed them in the cyanobacterial phylogeny and confirmed their genus membership. We then focused on the Nostocaceae family by concatenated analysis of four genes (16S rDNA, hetR, nifH, and rpoC1 genes) to characterize relationships among Anabaena morphospecies, in particular, Anabaena sphaerica var. tenuis G. S. West. Using a polyphasic approach to the Nostocaceae family, we demonstrate that A. sphaerica var. tenuis is more closely related to Cylindrospermospsis/Raphidiopsis than to other planktonic Anabaena/Aphanizomenon. On the basis of phylogeny and morphological data, we propose that these three significantly different clusters should be assigned to three genera.     

Nitrogen-fixing cyanobacterium <Emphasis Type="Italic">Trichormus variabilis</Emphasis> of the Lake Baikal phytoplankton

A new filamentous cyanobacterial strain BAC 9610 was isolated from the lake Baikal pelagial. Data obtained by light, scanning, and transmission electron microscopy, along with 16S rRNA gene sequence analysis, allowed the bacterium identification as Trichormus variabilis, previously known as Anabaena variabilis. Trichormus is a cyanobacterial genus not presented in the list of Baikal plankton algae; A. variabilis also hasn’t been previously detected in Baikal phytoplankton. T. variabilis nitrogen fixation ability was demonstrated. The gene responsible for nitrogen fixation, nifH, was identified by PCR and was partially sequenced. No hepatotoxin synthesis genes were revealed in the strain.     

Vindhyan Akinites: An Indicator of Mesoproterozoic Biospheric Evolution

A wide size range of rod-shaped, ellipsoidal akinites assignable to Archaeoellipsoides are reported from the Newari locality of the Mesoproterozoic Kheinjua Formation of the Semri Group, Vindhyan Supergroup. These akinites of heterocystous cyanobacteria (Archaeoellipsoides) represent the smallest of the forms reported from any other assemblage to date and are well comparable to the akinites of modern bloom forming Anabaena species. Like any other Mesoproterozoic microfossil assemblage, The Newari microfossil assemblage is also dominated by cyanobacterial population, but the presence of Archaeoellipsoides (akinites) or heterocyst forming Nostocales and Stigonematales are rather rarely reported. These fossils set a minimum date for the evolution of derived cyanobacteria, capable of marked cell differentiation, and they corroborate geochemical evidence indicating that atmospheric oxygen level was above 1% of present day level during Mesoproterozoic time. In the presence of oxygen a protected environment for nitrogenase (an oxygen sensitive nitrogen fixing enzyme) is produced by these akinites, which were abundant in coastal communities of Mesoproterozoic shallow marine carbonates. It is therefore interpreted that the presence of Vindhyan’s akinites indicate Mesoproterozoic biospheric evolution.     

Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.     

Detection and characterization of cyanobacterial nifH genes.

The DNA sequence of a 359-bp fragment of nifH was determined for the heterocystous strains Anabaena sp. strain CA (ATCC 33047), Nostoc muscorum UTEX 1933, a Nostoc sp., Gloeothece sp. strain ATCC 27152, Lyngbya lagerheimii UTEX 1930, and Plectonema boryanum IU 594. Results confirmed that the DNA sequence of the 359-bp segment is sufficiently variable to distinguish cyanobacterial nifH genes from other eubacterial and arachaeobacterial nifH genes, as well as to distinguish heterocystous from nonheterocystous nifH genes. Nonheterocystous cyanobacterial nifH sequences were greater than 70 and 82% identical on the DNA and amino acid levels, respectively, whereas corresponding values for heterocystous cyanobacterial nifH sequences were 84 and 91%. The amplified nifH fragments can be used as DNA probes to differentiate between species, although there was substantial cross-reactivity between the nifH amplification products of some strains. However, an oligonucleotide designed from a sequence conserved within the heterocystous cyanobacteria hybridized primarily with the amplification product from heterocystous strains. The use of oligonucleotides designed from amplified nifH sequences shows great promise for characterizing assemblages of diazotrophs.     

Rexia erecta Gen. et sp. nov. and Capsosira lowei sp. nov., Two Newly Described Cyanobacterial Taxa from the Great Smoky Mountains National Park (USA)

Two newly discovered taxa of Cyanobacteria from the Great Smoky Mountain National Park (USA) are presented. The first is the newly described species Capsosira lowei (Capsosiraceae), differing from the only other previously described species C. brebissonii Kütz. ex Born. et Flah. in regard to cell size and filament morphology. In addition, C. brebissonii is described as an aquatic or subaerophytic taxon, while our isolate was obtained as a phycobiont from the lichen Hydrothyria venosa J. L. Russell. Capsosira is currently placed in the Capsosiraceae of the Stigonematales due to its ability to have division in two planes. However, molecular evidence gathered in this study indicates closest affinity with Aulosira and Nostoc commune Vaucher, both in the Nostocaceae, Nostocales. Rexia erecta was isolated from concurrently collected aerophytic, epilithic sites. The hormogonia production, near absence of heterocysts and division in two planes are all typical of the Stigonematales, but it fits none of the currently circumscribed families in that order. This genus in other ways appears morphologically similar to members of the Scytonemataceae and Microchaetaceae. Molecular evidence (nearly complete 16S rRNA sequence data and 16S–23S internal transcribed spacer ITS region) places Rexia in the Microchaetaceae. These taxa are both problematic as they indicate that cell division in two planes has likely arisen more than once in the Nostocales, and thus the Stigonematales as currently circumscribed is not a monophyletic group. The Nostocales and Stigonematales are, in our opinion, in need of revision at the family and order level of classification.     

Fingerprinting and phylogeny of some heterocystous cyanobacteria using short tandemly repeated repetitive and highly iterated palindrome sequences

The presence of repeated DNA, viz. short tandemly repeated repetitive (STRR) and highly iterated palindrome (HIP) sequences was used as a typing technique for assessing genetic variability and phylogenetic relatedness of heterocystous cyanobacteria. Primers analogous to the STRR and HIP sequences were used to generate specific fingerprints for the twelve heterocystous cyanobacterial strains and a dendrogram was constructed. STRRmod and HIPTG primers revealed 100% polymorphism and yielded almost identical patterns. Anabaena sp. PCC 7120 clustered with Nostoc muscorum with both primers. Primer STRRmod supported the heterogeneity between Nostoc and Anabaena but HIPTG placed these two genera distinctly apart. STRRmod and HIPTG revealed that the members of the two orders were intermixed and thus suggesting a monophyletic origin of heterocystous cyanobacteria.     

Phylogeny and Characterization of Three nifH-Homologous Genes from Paenibacillus azotofixans

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.     

Nitrogen fixation andnif gene organization in branched heterocystous cyanobacteria: Variation in the presence ofxisA

Five branched heterocystous cyanobacteria (Scytonematopsis sp.,Scytonema sp.,Tolypothrix ceylonica, Mastigocladus sp. andFischerella sp.) were examined for their pattern of induction of nitrogenase activity andnif gene organization. All the forms showed the onset of nitrogenase activity after 12 h which could be correlated with the appearance of proheterocysts. The highest activity was exhibited byT. ceylonica. Hybridization studies revealed the presence of thenifD gene but the absence of thexisA gene inMastigocladus sp. andScytonematopsis sp. Interestingly,Tolypothrix sp. andScytonema sp. DNA samples hybridized withxisA. Hence no uniformity seems to exist regarding the presence ofxisA and the relatednif gene organization in branched heterocystous cyanobacteria. This investigation throws light on the primitive character and phylogenetic relatedness of branched forms to the coccoid/colonial forms. It also provides evidence for the proposition that stigonematacean cyanobacteria may not represent the most advanced cyanobacterial forms; rather they may link the coccoid and filamentous forms.