The Smallest Known Genomes of Multicellular and Toxic Cyanobacteria: Comparison,Minimal Gene Sets for Linked Traits and the Evolutionary Implications

Cyanobacterial morphology is diverse, ranging from unicellular spheres or rods to multicellular structures such as colonies and filaments. Multicellular species represent an evolutionary strategy to differentiate and compartmentalize certain metabolic functions for reproduction and nitrogen (N₂) fixation into specialized cell types (e.g. akinetes, heterocysts and diazocytes). Only a few filamentous, differentiated cyanobacterial species, with genome sizes over 5 Mb, have been sequenced. We sequenced the genomes of two strains of closely related filamentous cyanobacterial species to yield further insights into the molecular basis of the traits of N₂ fixation, filament formation and cell differentiation. Cylindrospermopsis raciborskii CS-505 is a cylindrospermopsin-producing strain from Australia, whereas Raphidiopsis brookii D9 from Brazil synthesizes neurotoxins associated with paralytic shellfish poisoning (PSP). Despite their different morphology, toxin composition and disjunct geographical distribution, these strains form a monophyletic group. With genome sizes of approximately 3.9 (CS-505) and 3.2 (D9) Mb, these are the smallest genomes described for free-living filamentous cyanobacteria. We observed remarkable gene order conservation (synteny) between these genomes despite the difference in repetitive element content, which accounts for most of the genome size difference between them. We show here that the strains share a specific set of 2539 genes with >90% average nucleotide identity. The fact that the CS-505 and D9 genomes are small and streamlined compared to those of other filamentous cyanobacterial species and the lack of the ability for heterocyst formation in strain D9 allowed us to define a core set of genes responsible for each trait in filamentous species. We presume that in strain D9 the ability to form proper heterocysts was secondarily lost together with N₂ fixation capacity. Further comparisons to all available cyanobacterial genomes covering almost the entire evolutionary branch revealed a common minimal gene set for each of these cyanobacterial traits.     

Timing of morphological and ecological innovations in the cyanobacteria – a key to understanding the rise in atmospheric oxygen

When cyanobacteria originated and diversified, and what their ancient traits were, remain critical unresolved problems. Here, we used a phylogenomic approach to construct a well‐resolved ‘core’ cyanobacterial tree. The branching positions of four lineages (Thermosynechococcus elongatus, Synechococcus elongatus, Synechococcus PCC 7335 and Acaryochloris marina) were problematic, probably due to long branch attraction artifacts. A consensus genomic tree was used to study trait evolution using ancestral state reconstruction (ASR). The early cyanobacteria were probably unicellular, freshwater, had small cell diameters, and lacked the traits to form thick microbial mats. Relaxed molecular clock analyses suggested that early cyanobacterial lineages were restricted to freshwater ecosystems until at least 2.4 Ga, before diversifying into coastal brackish and marine environments. The resultant increases in niche space and nutrient availability, and consequent sedimentation of organic carbon into the deep oceans, would have generated large pulses of oxygen into the biosphere, possibly explaining why oxygen rose so rapidly. Rapid atmospheric oxidation could have destroyed the methane‐driven greenhouse with simultaneous drawdown in pCO₂, precipitating ‘Snowball Earth’ conditions. The traits associated with the formation of thick, laminated microbial mats (large cell diameters, filamentous growth, sheaths, motility and nitrogen fixation) were not seen until after diversification of the LPP, SPM and PNT clades, after 2.32 Ga. The appearance of these traits overlaps with a global carbon isotopic excursion between 2.2 and 2.1 Ga. Thus, a massive re‐ordering of biogeochemical cycles caused by the appearance of complex laminated microbial communities in marine environments may have caused this excursion. Finally, we show that ASR may provide an explanation for why cyanobacterial microfossils have not been observed until after 2.0 Ga, and make suggestions for how future paleobiological searches for early cyanobacteria might proceed. In summary, key evolutionary events in the microbial world may have triggered some of the key geologic upheavals on the Paleoproterozoic Earth.     

Cellular differentiation in the cyanobacterium Nostoc punctiforme

Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.     

Chroococcidiopsis and heterocyst-differentiating cyanobacteria are each other's closest living relatives

Many filamentous cyanobacteria reduce atmospheric nitrogen in specialized differentiated cells called heterocysts. Here we present evidence that shows that members of the unicellular non-heterocyst-differentiating genus Chroococcidiopsis and the filamentous heterocyst-differentiating cyanobacteria are each other's closest living relatives. Distance, maximum-parsimony, and maximum-likelihood analyses of complete small subunit ribosomal RNA gene sequences yielded highly congruent support for the monophyly of Chroococcidiopsis and the heterocyst-differentiating cyanobacteria. Our results demonstrate that the order Pleurocapsales, which traditionally contains Chroococcidiopsis, is a polyphyletic assemblage with the ability to reproduce by multiple fission having arisen independently at least twice during the cyanobacterial radiation. Our data also reject Myxosarcina as a sister taxon to Chroococcidiopsis, indicating that the numerous presumed shared derived characters thought to unite the two genera evolved independently. The sequence divergence within the Chroococcidiopsis lineage is comparable to and probably exceeds that in the entire heterocyst-differentiating lineage. Chroococcidiopsis forms unique survival cells under nitrogen-limiting conditions, and the sister group relationship with the heterocystous cyanobacteria shown here suggests that differentiation of these cells and heterocysts may be related processes.     

Molecular phylogeny of the Cladophoraceae (Cladophorales,Ulvophyceae), with the resurrection of Acrocladus Nägeli and Willeella Børgesen,and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov.

The taxonomy of the Cladophoraceae, a large family of filamentous green algae, has been problematic for a long time due to morphological simplicity, parallel evolution, phenotypic plasticity, and unknown distribution ranges. Partial large subunit (LSU) rDNA sequences were generated for 362 isolates, and the analyses of a concatenated dataset consisting of unique LSU and small subunit (SSU) rDNA sequences of 95 specimens greatly clarified the phylogeny of the Cladophoraceae. The phylogenetic reconstructions showed that the three currently accepted genera Chaetomorpha, Cladophora, and Rhizoclonium are polyphyletic. The backbone of the phylogeny is robust and the relationships of the main lineages were inferred with high support, only the phylogenetic position of both Chaetomorpha melagonium and Cladophora rupestris could not be inferred unambiguously. There have been at least three independent switches between branched and unbranched morphologies within the Cladophoraceae. Freshwater environments have been colonized twice independently, namely by the freshwater Cladophora species as well as by several lineages of the Rhizoclonium riparium clade. In an effort to establish monophyletic genera, the genera Acrocladus and Willeella are resurrected and two new genera are described: Pseudorhizoclonium and Lurbica.     

MOLECULAR CHARACTERIZATION OF THE DIVERSITY AND POTENTIAL TOXICITY OF CYANOBACTERIAL MATS IN TWO TROPICAL LAGOONS IN THE SOUTH PACIFIC OCEAN1

Marine benthic cyanobacteria in tropical areas have recently been associated with several human poisoning events. To enhance the characterization of these microorganisms and their potential toxicity, benthic cyanobacterial communities were sampled in the lagoons of two islands (Raivavae and Rurutu) located in French Polynesia where human poisoning events by seafood had been reported. The morphological appearance of the mats was used to identify four types of cyanobacterial mat. By a 16S rRNA sequencing approach, it appeared that these mats were usually dominated by a restricted number of operational taxonomic units (OTUs), which were closely related to Leptolyngbya, Oscillatoria, Hydrocoleum, and Anabaena sequences, as previously reported in other tropical lagoons. Interestingly, we determined that these dominant filamentous OTUs were associated in the mats with other cyanobacteria, including unicellular species. By using a population genetic approach based on the sequencing of the internally transcribed spacer (ITS) of the rRNA operon, we found a very restricted genetic diversity in the most common OTU, which displayed a high sequence similarity with Leptolyngbya sp. In addition, there was no geographic differentiation at various spatial scales in the distribution of the different genotypes, suggesting that this species is able to spread over large distances. Finally, PCR screening of genes involved in the biosynthesis of known cyanotoxins revealed the presence of the saxitoxin gene (stxG) in two mats containing a mix of filamentous and unicellular cyanobacterial species.     

Morphological and molecular characterization of <Emphasis Type="Italic">Calothrix</Emphasis> isolates obtained from diverse environments in India

Thirty cyanobacterial strains of Calothrix (family Rivulariaceae) isolated from diverse geographical regions of India were analyzed using morphological and molecular approaches. Most of the isolates were planktonic while some grew benthically. Significant differences were observed with regard to the shape and size of the vegetative cells, heterocysts, and akinetes. Analyses of molecular polymorphisms using Restriction Fragment Length Polymorphisms (RPLP) of 16S rRNA genes with the reference strain led to unambiguous differentiation of the isolates as well as understanding of their genetic relationships.     

Community phylogenetic analysis of moderately thermophilic cyanobacterial mats from China, the Philippines and Thailand

Most community molecular studies of thermophilic cyanobacterial mats to date have focused on Synechococcus occurring at temperatures of ~50–65°C. These reveal that molecular diversity exceeds that indicated by morphology, and that phylogeographic lineages exist. The moderately thermophilic and generally filamentous cyanobacterial mat communities occurring at lower temperatures have not previously been investigated at the community molecular level. Here we report community diversity in mats of 42–53°C recovered from previously unstudied geothermal locations. Separation of 16S rRNA gene-defined genotypes from community DNA was achieved by DGGE. Genotypic diversity was greater than morphotype diversity in all mats sampled, although genotypes generally corresponded to observed morphotypes. Thirty-six sequences were recovered from DGGE bands. Phylogenetic analyses revealed these to form novel thermophilic lineages distinct from their mesophilic counterparts, within Calothrix, Cyanothece, Fischerella, Phormidium, Pleurocapsa, Oscillatoria and Synechococcus. Where filamentous cyanobacterial sequences belonging to the same genus were recovered from the same site, these were generally closely affiliated. Location-specific sequences were observed for some genotypes recovered from geochemically similar yet spatially separated sites, thus providing evidence for phylogeographic lineages that evolve in isolation. Other genotypes were more closely affiliated to geographically remote counterparts from similar habitats suggesting that adaptation to certain niches is also important.     

Cyanobacteria-bryophyte symbioses

Cyanobacteria are a large group of photosynthetic prokaryotesof enormous environmental importance, being responsible fora large proportion of global CO₂ and N₂ fixation. They formsymbiotic associations with a wide range of eukaryotic hostsincluding plants, fungi, sponges, and protists. The cyanobacterialsymbionts are often filamentous and fix N₂ in specialized cellsknown as heterocysts, enabling them to provide the host withfixed nitrogen and, in the case of non-photosynthetic hosts,with fixed carbon. The best studied cyanobacterial symbiosesare those with plants, in which the cyanobacteria can infectthe roots, stems, leaves, and, in the case of the liverwortsand hornworts, the subject of this review, the thallus. Thesymbionts are usually Nostoc spp. that gain entry to the hostby means of specialized motile filaments known as hormogonia.The host plant releases chemical signals that stimulate hormogoniaformation and, by chemoattraction, guide the hormogonia to thepoint of entry into the plant tissue. Inside the symbiotic cavity,host signals inhibit further hormogonia formation and stimulateheterocyst development and dinitrogen fixation. The cyanobiontsundergo morphological and physiological changes, including reducedgrowth rate and CO₂ fixation, and enhanced N₂ fixation, andrelease to the plant much of the dinitrogen fixed. This shortreview summarizes knowledge of the cyanobacterial symbioseswith liverworts and hornworts, with particular emphasis on theimportance of pili and gliding motility for the symbiotic competenceof hormogonia. Key words: Bryophyte, cyanobacteria, gliding motility, pili, symbiosis Received 1 August 2007; Revised 23 December 2007 Accepted 7 January 2008     

Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae)

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.     

A NUMERICAL TAXONOMIC STUDY OF NOSTOC AND ANABAENA1

Thirty characteristics of 14 Nostoc and 10 Anabaena species were analyzed from previously published data. Using standard numerical taxonomic methods, simple matching coefficients were calculated and a phenogram drawn. The analysis revealed that some of the central characteristics of Nostoc are: a punctiforme stage; motile reproductive stage; plant mass with a dull to shiny luster, non-veined surface, and nonfimbriate margin; some spherical vegetative cells; no cylindrical heterocysts; and some spherical, but no cylindrical akinetes. Some of the central characteristics of Anabaena that were revealed are: no punctiforme stage; a motile vegetative stage; plant mass with a shiny luster, veined surface, and fimbriate margin; no spherical vegetative cells; some cylindrical heterocysts; and some cylindrical, but no spherical, akinetes. In general, Anabaena has larger akinetes and vegetative cells than Nostoc. Based on 30 morphological characteristics and the clustering data of the phenogram, keys were constructed for the Nostoc and Anabaena species studied. The data clearly support two separate and distinct, though similar genera and, less sharply, the separation of the 24 species. The more useful characteristics for separation of the species are size and shape of akinetes, vegetative cells, and heterocysts; color and luster of plant mass; veined plant mass surface; margin fimbriate; and shape of plant mass in nature.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Detection of Seven Major Evolutionary Lineages in Cyanobacteria Based on the 16S rRNA Gene Sequence Analysis with New Sequences of Five Marine Synechococcus Strains

Although molecular phylogenetic studies of cyanobacteria on the basis of the 16S rRNA gene sequence have been reported, the topologies were unstable, especially in the inner branchings. Our analysis of 16S rRNA gene phylogeny by the maximum-likelihood and neighbor-joining methods combined with rate homogeneous and heterogeneous models revealed seven major evolutionary lineages of the cyanobacteria, including prochlorophycean organisms. These seven lineages are always stable on any combination of these methods and models, fundamentally corresponding to phylogenetic relationships based on other genes, e.g., psbA, rbcL, rnpB, rpoC, and tufA. Moreover, although known genotypic and phenotypic characters sometimes appear paralleled in independent lineages, many characters are not contradictory within each group. Therefore we propose seven evolutionary groups as a working hypothesis for successive taxonomic reconstruction. New 16S rRNA sequences of five unicellular cyanobacterial strains, PCC 7001, PCC 7003, PCC 73109, PCC 7117, and PCC 7335 of Synechococcus sp., were determined in this study. Although all these strains have been assigned to ``marine clusters B and C,' they were separated into three lineages. This suggests that the organisms classified in the genus Synechococcus evolved diversely and should be reclassified in several independent taxonomic units. Moreover, Synechococcus strains and filamentous cyanobacteria make a monophyletic group supported by a comparatively high statistical confidence value (80 to 100%) in each of the two independent lineages; therefore, these monophylies probably reflect the convergent evolution of a multicellular organization. Received: 3 September 1998 / Accepted: 30 November 1998     

Cyanobacteria of Rocks and Soils of the Orinoco Lowlands and the Guayana Uplands,Venezuela

The occurrence of 23 cyanobacterial species, belonging to 9 different genera and 5 cyanobacterial lichen species of 5 different genera on exposed, open rock surfaces of inselbergs and on soil in savannas of the Orinoco lowlands and the Guayana uplands is described. Their distribution patterns and frequency within the different habitats are given. The filamentous procaryotic blue-green algae/cyanobacteria Stigonema ocellatum and Scytonema crassum, together with the unicellular cyanobacterium Gloeocapsa sanguinea were the most frequent species on rocks, whereas the filamentous cyanobacterium, Schizothrix telephoroides, dominated in cyanobacterial mats on the savanna soil. All species showed intensively coloured sheaths, either brown or yellow in the case of Stigonema ocellatum and Scytonema crassum, or red in Gloeocapsa sanguinea and Schizothrix telephoroides. In addition, a number of cyanobacterial lichens occurred.     

Motility in cyanobacteria: polysaccharide tracks and Type IV pilus motors

Motility in cyanobacteria is useful for purposes that range from seeking out favourable light environments to establishing symbioses with plants and fungi. No known cyanobacterium is equipped with flagella, but a diverse range of species is able to ‘glide’ or ‘twitch’ across surfaces. Cyanobacteria with this capacity range from unicellular species to complex filamentous forms, including species such as Nostoc punctiforme, which can generate specialised motile filaments called hormogonia. Recent work on the model unicellular cyanobacterium Synechocystis sp. PCC 6803 has shown that its means of propulsion has much in common with the twitching motility of heterotrophs such as Pseudomonas and Myxococcus. Movement depends on Type IV pili, which are extended, adhere to the substrate and then retract to pull the cell across the surface. Previous work on filamentous cyanobacteria suggested a very different mechanism, with movement powered by the directional extrusion of polysaccharide from pores close to the cell junctions. Now a new report by Khayatan and colleagues in this issue of Molecular Microbiology suggests that the motility of Nostoc hormogonia has much more in common with Synechocystis than was previously thought. In both cases, polysaccharide secretion is important for preparing the surface, but the directional motive force comes from Type IV pili.