Figure 1.
The partners in the Azolla symbiosis.
A) Fronds of the Azolla filiculoides Lam. plant. B) Close up of an Azolla branch showing the apex and the alternating ‘stacked’ dorsal leaves, each containing a cavity in which the cyanobiont (NoAz) filaments reside. C) Light micrograph of the cyanobiont. The larger cells in the vegetative filaments represent the nitrogen-fixing heterocysts. Scale bar = 5 µm. D) Transmission electron micrograph of the cyanobiont. Note the thicker cell-walls and the electron dense polar nodules of the heterocyst (middle cell) at the interface to flanking vegetative cells, which function as combined N storage structures (cyanophycin). Scale bar = 5 µm E) A snap-shot in the vertical transmission process of the cyanobiont between Azolla plant generations, using fluorescence microscopy. Pairs of megasporocarps (blue) develop at the underside of the cyanobacterial colonized Azolla leaves. Filaments of the motile cyanobacterial cell stage (red), the hormogonia (h), are attracted to the sporocarps, gather at the base and subsequently move towards the tip, before entering the sporocarps via channels (white arrows). Once inside the sporocarp the hormogonia differentiate into individual thick walled resting spores (or akinetes; ak), seen as the intensively red fluorescing small inoculum on top of the megaspores (sp). For details see [15].
Figure 2.
Phylogenetic tree and genome sizes for ten filamentous cyanobacterial species.
The closest relatives to ‘Nostoc azollae’ 0708 are Raphidiopsis brookii D9 and Cylindrospermopsis raciborskii CS 505, the two multicellular cyanobacteria with the smallest known genomes. The tree is a subclade from a maximum likelihood analysis of all cyanobacterial genomes available from NCBI and IMG/ER (see Material and Methods).
Figure 3.
Map of the main chromosome, and plasmids (P1, P2) of the ‘Nostoc azollae’ 0708 genome.
The distribution of pseudogenes (red) and remains of insertion elements (blue) are indicated. Predicted genes are indicated by grey color. The highest level of gene erosion (number of pseudogenes:number of predicted genes) is found in the plasmid P1. Note that the occurrence of insertion elements appears to be correlated with the distribution of pseudogenes. The P1 and P2 plasmids only contain two and one remains of insertion elements, respectively.
Table 1.
Overview of genome features in the cyanobiont (‘Nostoc azollae’ 0708) of the water fern Azolla filiculoides Lam.
Figure 4.
Classification of genes in COG functional categories.
A) Distribution of genes and pseudogenes in functional categories. Percentages signify the amount of pseudogenes in each category. A Pearson's Chi-squared test (see Materials and Methods) shows that the distribution of pseudogenes within COG categories is non-random. B) Residuals from the Pearson's Chi-squared test. Large positive values indicates a stronger overrepresentation of pseudogenes while large negative values indicate stronger underrepresentation. (B) = Chromatin structure and dynamics, (C) = Energy production and conversion, (D) = Cell cycle control, cell division, chromosome partitioning, (E) = Amino acid transport and metabolism, (F) = Nucleotide transport and metabolism, (G) = Carbohydrate transport and metabolism, (H) = Coenzyme transport and metabolism, (I) = Lipid transport and metabolism, (J) = Translation, ribosomal structure and biogenesis, (K) = Transcription, (L) = Replication, recombination and repair, (M) = Cell wall/membrane/envelope biogenesis, (N) = Cell motility, (O) = Posttranslational modification, protein turnover, chaperones, (P) = Inorganic ion transport and metabolism, (Q) = Secondary metabolites biosynthesis, transport and catabolism, (R) = General function prediction only, (S) = Function unknown, (T) = Signal transduction mechanisms, (U) = Intracellular trafficking, secretion, and vesicular transport, (V) = Defense mechanisms.
Figure 5.
Examples of gene fragmentation in ‘Nostoc azollae’ 0708 (NoAz) compared to other cyanobacteria.
Nostoc punctiforme PCC 73102 (Nosp) and Nostoc sp. PCC 7120 (Noss). Best reciprocal BLAST hits between genomes are indicated for each image subset by numbers in parenthesis below genes. Transposases are seen in red. Pseudogenes are indicated by the * suffix. Gaps in the sequence are indicated by three dots and the length of the omitted sequence. A) The dnaA region. Vertical black arrows indicate oriC regions predicted by Ori-Finder (see Materials and Methods). Note the fragmentation of the dnaA gene and the putative transposase between dnaA and dnaN in NoAz. Although large genomic parts appear to have been lost from the NoAz genome, the organization of several genes in the different species is conserved. B) A cluster of genes involved in galactose/polysaccharide metabolism. This gene cluster is not present in any other cyanobacterial genome in the IMG database. Note that genes in NoAz are heavily fragmented in comparison to Nosp and that the gene organization is rearranged. Transposases are present between ORFs and also within the UDP-galactopyranose mutase gene in NoAz. The genes encoding transposases are also fragmented.
Figure 6.
Schematic illustration of important metabolic and genetic information pathways in NoAz.
The left cell represents a vegetative cell while the right a nitrogen-fixing heterocyst. Red color indicates pseudogenes lacking a functional counterpart in the NoAz genome. Orange indicates pseudogenes where a functional counterpart is present elsewhere in the genome. Fully functional gene(s) are illustrated (blue) only if their function is linked to other processes in the figure. The localization of pathways in vegetative cells or heterocysts is representative only for nitrogen fixation (heterocysts) and PSII activity (vegetative cells). Note that only a minor part of the nitrogen fixed in heterocysts is incorporated using the GS-GOGAT pathway and used for synthesis of amino acids, while most is exported to the plant as NH3. Sugar is provided by the plant in an as yet unknown form; putatively imported via the sugar phosphotransferase system (PTS). Function has been lost in the glycolytic pathway as the pfkA gene, encoding 6-phosphofructokinase, is a pseudogene and sugar metabolism in the Azolla cyanobiont probably proceeds via the Oxidative Pentose Phosphate Pathway (OPPP). Extensive loss of function is evident among genes involved in uptake and transport of nutrients and NoAz has lost the capacity to both import and metabolise alternative nitrogen sources. Table S3 shows detailed information on genes indicated in the figure and their closest homologs in other filamentous heterocystous cyanobacteria.
Figure 7.
Illustration of genes related to N2-fixation, a highly conserved gene cluster in cyanobacteria.
The structural genes for the nitrogenase enzyme (nifHDK) are highlighted in color for clarity. Also, genes which differ in terms of occurrence and/or organization are indicated in grey. The nitrogenase enzyme catalyzes the fixation of atmospheric dinitrogen gas. Transposases are indicated in red. Three dots indicate gaps and incision elements, with the length of the omitted sequence given.