Complete genome sequence of Anabaena variabilis ATCC 29413
© The Author(s) 2014
Published: 15 June 2014
Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40° C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.
Anabaena variabilis ATCC 29413 (=IUCC 1444 = PCC 7937) is a semi-thermophilic, filamentous, heterocyst-forming cyanobacterium. Heterocysts, which are specialized cells that form in a semi-regular pattern in the filament, are the sites of nitrogen fixation in cells grown in an oxic environment. A. variabilis ATCC 29413 was first isolated as a freshwater strain in 1964 in Mississippi by R.G. Tischer, who called the strain Anabaena flos-aquae A-37 . He was primarily interested in the extracellular polysaccharide produced by this strain [2–4], which was subsequently called Anabaena variabilis by Healey in 1973 . It was characterized in more detail by several labs in the 1960’s and 1970’s [6–8]. In particular, the early work by Wolk on this strain led to its becoming a model strain for cyanobacterial physiology, nitrogen fixation and heterocyst formation [9–13]. Here we present a summary classification and a set of features for A. variabilis ATCC 29413 together with the description of the complete genomic sequencing and annotation.
Classification and features
Classification and general features of A. variabilis ATCC 9413 according to the MIGS recommendations 
Species Anabaena variabilis
Ovoid cells in filaments
20 – 40°C
Relationship to Oxygen
TAS (this report)
TAS (this report)
Free living; symbiotic
Isolated Mississippi, 1964
A. variabilis is a well-established model organism for heterocyst formation [35,36], nitrogen fixation [21,37,38], hydrogen production [39,40], photosynthesis [41–43], and heterotrophic cyanobacterial growth [9,32,44]. It is unique among the well-characterized cyanobacteria in that it has three sets of genes that encode distinct nitrogenases [19,37,38,45–48]. One is the conventional, heterocyst-specific Mo-nitrogenase, the second is another Mo-nitrogenase that functions only under anoxic conditions in vegetative cells and heterocysts, while the third is a V-nitrogenase that is also heterocyst specific. These nitrogenases are expressed under distinct physiological conditions so that only one nitrogenase is generally functional . The genome sequence has revealed a large 41-kb island of genes that all appear to be involved in synthesis and regulation of the V-nitrogenase, including the genes for the first vanadate transport system to be characterized in any bacterium . The V-nitrogenase of A. variabilis has been exploited for its ability to make large amounts of hydrogen as a potential source of alternative energy production [39,40].
The Gram-negative cyanobacterial cell wall has not been well characterized; however, it typically contains lipopolysaccharide. In A. variabilis the O antigen contains L-acofriose, L-rhamnose, D-mannose, D-glucose, and D-galactose . The cell envelope of the heterocyst differs from vegetative cells in that it also contains an inner laminated glycolipid layer and an outer fibrous, homogeneous polysaccharide layer. In A. variabilis the polysaccharide layer comprises a 1,3-linked backbone of glucosyl and mannosyl residues with terminal xylosyl and galactosyl residues. The side branches comprise glucosyl residues having a terminal arabinosyl residue. The inner heterocyst cell wall of almost all strains of Anabaena and Nostoc consists of a glycolipid comprising 1-(O-hexose)-3,25-hexacosanediol and 1-(O-hexose)-3-keto-25-hexacosanol [51,52]. The lipids of most cyanobacteria comprise monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulphoquinovosyldiacylglycerols and phosphatidylglycerols .
In A. variabilis the primary products of lipid biosynthesis are 1-stearoyl-2-palmitoyl species of monoglucosyl diacylglycerol, phosphatidylglycerol and sulfoquinovosyl diacylglycerol; however, the degree of saturation of the fatty acids in the lipids depends on the growth temperature [54–56]
Genome project history
Finished - < one error per 50 kb
3-kb pUC18c; 9-kbpMCL200; 40-kb pCC1Fos
Gene calling method
Genbank Date of Release
September 17, 2005
Hydrogen production; nitrogen fixation
The strain was first isolated by R.G. Tischer, in 1964 in Mississippi, who called it Anabaena flos-aquae MSU A-3 7 . It was submitted to the Indiana University Culture Collection (Anabaena flos-aquae IUCC 1444) and was then submitted by C.P Wolk as Anabaena variabilis to ATCC in 1976 (Anabaena variabilis ATCC 29413). The phylogenetic tree (Figure 1) reveals that the strain clusters with cyanobacteria in the genus Nostoc, which is consistent with the fact that it produces hormogonia , and not with the cluster of Anabaena/Aphanizomenon, suggests that the strain was incorrectly named.
Growth conditions and DNA isolation
An axenic culture of A. variabilis ATCC 29413 was grown photoautotrophically in one L of an eight-fold dilution of the medium of Allen and Arnon (AA/8) , supplemented 5.0 mM NaNO3 at 30°C with illumination at 50–80 µ Einsteins m−2 s−1 to an OD720 of about 0.3. Cells were harvested by centrifugation, frozen and then lysed by a combination of crushing the frozen pellet with a very cold mortar and pestle, and then treating the frozen powder with lysozyme (3.0 mg/ml)/proteinase K (1 mg/ml) in 10 mM Tris, 100 mM EDTA pH 8.0 buffer at 37°C for 30 min. This was followed by purification of the DNA using a Qiagen genomic DNA kit. The DNA was precipitated with isopropanol, spooled, and then dissolved in 10 mM Tris, 1.0 mM EDTA pH 8.0 buffer. The purity, quality and size of the bulk gDNA preparation were assessed by JGI according to DOE-JGI guidelines.
Genome sequencing and annotation
Sequencing and assembly
Sanger sequencing was done using a whole-genome shotgun approach with three plasmid libraries. A pUC18c library with 3-kb inserts generated 39.64 Mb of sequence. A pMCL200 library with 9-kb inserts produced 35.16 Mb of sequence, and a fosmid(pCC1Fos CopyControl fosmid library production kit; Epicentre, Madison, WI) library with 40-kb inserts yielded 5.83 Mb of sequence. Together, all libraries provided greater than 11.0× coverage of the genome. The plasmid inserts were made with sheared DNA that was blunt-end repaired and then size separated by gel electrophoresis. Sequencing from both ends of the plasmid inserts was done using dye terminators on ABI3730 sequencers. Details on the cloning and sequencing procedures are available from JGI . Project information is summarized in Table 2.
The Phred, Phrap, and Consed software package was used for sequence assembly and quality assessment  Repeat sequences were resolved with Dupfinisher . Gaps between contigs were closed by editing in Consed, custom priming, or PCR amplification. This genome was curated to close all gaps with greater than 98% coverage of at least two independent clones. Each base pair has a minimum q (quality) value of 30 and the total error rate is less than one per 50,000.
Genes were identified using two gene modeling programs, Glimmer  and Critica  as part of the Oak Ridge National Laboratory genome annotation pipeline .The two sets of gene calls were combined using Critica as the preferred start call for genes with the same stop codon. Genes with less than 80 amino acids that were predicted by only one of the gene callers and had no Blast hit in the KEGG database at 1e−5 were deleted. This was followed by a round of manual curation to eliminate obvious overlaps. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE , TMHMM , and signal .
Summary of genome: one chromosome, three plasmids and one linear element
Nucleotide content and gene count levels of the genome
% of total
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Protein coding genes with function prediction
Genes in paralog clusters
Genes assigned to COGs
Genes assigned Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Identification of vnf genes in other cyanobacterial genomes
A. variabilis was one of the earliest model organisms for the study of important cellular processes such as photosynthesis and nitrogen fixation. It is unusual among cyanobacteria in that it has three nitrogenases , one of which, the V-nitrogenase, has been shown to be useful for hydrogen production , and for its ability to grow both photoautotrophically in the light and heterotrophically in the dark. The genome sequence was critical in identifying the genes for fructose transport  and the large island of genes important for V-nitrogenase function, including the vupABC genes for vanadate transport . No other cyanobacterial genome has all the genes identified in A. variabilis that are important for growth using the V-nitrogenase, but two strains, Fischerella 9339 and Chlorogleopsis 7702, have some V-nitrogenase or vanadate transport genes. The presence of the linear genetic element shown in Fig. 4 is quite interesting, as such elements are not present in the genomes of the other Nostoc strains. It will also be interesting to determine whether this element is important to the cell and how this element replicates.
Support for this research was provided to Teresa Thiel by National Science Foundation grant MCB-1052241. The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under contract No. DE-AC02-05CH11231.
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