Novel Synechococcus Genomes Reconstructed from Freshwater Reservoirs

Abstract

Freshwater picocyanobacteria including Synechococcus remain poorly studied at the genomic level, compared to their marine representatives. Here, using a metagenomic assembly approach we discovered two novel Synechococcus sp. genomes from two freshwater reservoirs Tous and Lake Lanier, both sharing 96% average nucleotide identity and displaying high abundance levels in these two lakes located at similar altitudes and temperate latitudes. These new genomes have the smallest estimated size (2.2 Mb) and average intergenic spacer length (20 bp) of any previously sequenced freshwater Synechococcus, which may contribute to their success in oligotrophic freshwater systems. Fluorescent in situ hybridization confirmed that Synechococcus sp. Tous comprises small cells (0.987 ± 0.139 μm length, 0.723 ± 0.119 μm width) that amount to 90% of the picocyanobacteria in Tous. They appear together in a phylogenomic tree with Synechococcus sp. RCC307 strain, the main representative of sub-cluster 5.3 that has itself one of the smallest marine Synechococcus genomes. We detected a type II phycobilisome (PBS) gene cluster in both genomes, which suggests that they belong to a phycoerythrin-rich pink low-light ecotype. The decrease of acidic proteins and the higher content of basic transporters and membrane proteins in the novel Synechococcus genomes, compared to marine representatives, support their freshwater specialization. A sulfate Cys transporter which is absent in marine but has been identified in many freshwater cyanobacteria was also detected in Synechococcus sp. Tous. The RuBisCo subunits from this microbe are phylogenetically close to the freshwater amoeba Paulinella chromatophora symbiont, hinting to a freshwater origin of the carboxysome operon of this protist. The novel genomes enlarge the known diversity of freshwater Synechococcus and improve the overall knowledge of the relationships among members of this genus at large.

Keywords: Synechococcus; abundance; freshwater reservoirs; metagenomics; picocyanobacteria; smallest estimated size.

FIGURE 1

(A) Phylogenomics of the genus Synechococcus sp. One hundred twenty-two conserved genes were used to generate a maximum-likelihood phylogenetic tree with marine, freshwater and the novel Synechococcus sp. representatives. Three Prochlorococcus genomes were used as an outgroup. (B) ANI (average nucleotide identity) between the closest marine and freshwater representatives to the novel Synechococcus sp. Tous/Lanier.

FIGURE 2

(A) Genomic comparison between marine Synechococcus sp. RCC307 and the novel freshwater assembled genomes from Tous and Lanier. Comparison made with BLASTN with 50 bp alignment length and >70% similarity. 16S rRNA, DnaA replication initiator, carboxysome operon, Cys sulfate transporter and PBS genes are indicated in the genomes. (B) Genome size (Mbp) vs. GC content of all marine, freshwater and euryhaline Synechococcus sp. sequenced genomes. (C) Genome size (Mbp) vs. median intergenic spacer (bp) of all Synechococcus sp. genomes. Estimated genome size was used for the novel freshwater Synechococcus sp.

FIGURE 3

(A) Fragment recruitment of novel and reference Synechococcus sp. Genome abundance (expressed as RPKG, reads per Kb of genome per Gb of metagenome) along different freshwater metagenomes and two marine metagenomes. Only datasets with >5 RPKG values for the novel freshwater Synechococcus sp. were included in this analysis. Only hits with ≥95% identity, ≥50 bp alignment length were considered. Sep, September; Nov, November; Feb, February; Jul, July; Aug, August; SF, small fraction. (B) Metagenomic recruitment of Synechococcus sp. Tous and Lanier assembled genomes against Lanier S2 sample (5 m August 28th, 2011) (I) and Tous 12 m February 2014 (II) metagenomes. A 95% identity cut-off and 50 bp read length on each metagenome were used as restrictive parameters.

FIGURE 4

Photomicrographs (1250×) of a 12 m depth sample from Tous reservoir. (A) Phycoerythrin fluorescence under a green filter showing all picocyanobacteria in the sample. (B) FISH microphotograph of the same microscopic field stained with the Cy3, Cy5 fluorescence labeled rRNA-targeted probe specific for Synechococcus sp. Tous (“SynTo”). Note that all picocyanobacteria except that marked by the arrow are targeted by the specific “SynTous” probe. Scale bars represent 10 μm.

FIGURE 5

Structure and similarity of the PBS gene cluster among marine and Tous/Lanier freshwater Synechococcus sp. Comparison made with TBLASTX with >30% similarity hits and 150 bp alignment lengths. PC, Phycocyanin; PE, Phycoerythrin; AplA, Allophycoyanin-like protein; Phycobilin lyases, CpeY, CpeZ, CpeF, CpeS, CpeT, CpeU, RpcG, RpcE, RpcE, RpcF, RpcT; Bilin synthesis, pebA, pebB.