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.

Cavanilles Institute of Biodiversity and Evolutionary Biology University of ValenciaValencia Spain

Evolutionary Genomics Group Departamento de Producción Vegetal y Microbiología Universidad Miguel HernándezSan Juan de Alicante Spain

Institute of Hydrobiology Department of Aquatic Microbial Ecology Biology Center of the Academy of Sciences of the Czech RepublicČeské Budějovice Czechia

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Albertsen M., Hugenholtz P., Skarshewski A., Nielsen K. L., Tyson G. W., Nielsen P. H. (2013). Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nat. Biotechnol. 31 533–538. 10.1038/nbt.2579 PubMed DOI

Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 3389–3402. 10.1093/nar/25.17.3389 PubMed DOI PMC

American Public Health Association, American Water Works Association, World Powerlifting Congress Federation and Water Environmental Federation (1915). Standard Methods for the Examination of Water and Wastewater. Washington, DC: American Public Health Association.

Arnosti C., Fuchs B. M., Amann R., Passow U. (2012). Contrasting extracellular enzyme activities of particle-associated bacteria from distinct provinces of the North Atlantic Ocean. Front. Microbiol. 3:425 10.3389/fmicb.2012.00425 PubMed DOI PMC

Bankevich A., Nurk S., Antipov D., Gurevich A. A., Dvorkin M., Kulikov A. S., et al. (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19 455–477. 10.1089/cmb.2012.0021 PubMed DOI PMC

Bardavid R. E., Oren A. (2012). Acid-shifted isoelectric point profiles of the proteins in a hypersaline microbial mat: an adaptation to life at high salt concentrations? Extremophiles 16 787–792. 10.1007/s00792-012-0476-6 PubMed DOI

Behnam F., Vilcinskas A., Wagner M., Stoecker K. (2012). A straightforward DOPE (double labeling of oligonucleotide probes)-FISH (fluorescence in situ hybridization) method for simultaneous multicolor detection of six microbial populations. Appl. Environ. Microbiol. 78 5138–5142. 10.1128/AEM.00977-12 PubMed DOI PMC

Bhaya D., Grossman A. R., Steunou A.-S., Khuri N., Cohan F. M., Hamamura N., et al. (2007). Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses. ISME J. 1 703–713. 10.1038/ismej.2007.46 PubMed DOI

Billini M., Stamatakis K., Sophianopoulou V. (2008). Two members of a network of putative Na+/H+ antiporters are involved in salt and pH tolerance of the freshwater cyanobacterium Synechococcus elongatus. J. Bacteriol. 190 6318–6329. 10.1128/JB.00696-08 PubMed DOI PMC

Blank C., Sanchez-Baracaldo P. (2010). Timing of morphological and ecological innovations in the cyanobacteria–a key to understanding the rise in atmospheric oxygen. Geobiology 8 1–23. 10.1111/j.1472-4669.2009.00220.x PubMed DOI

Blindauer C. A. (2008). Zinc-handling in cyanobacteria: an update. Chem. Biodivers. 5 1990–2013. 10.1002/cbdv.200890183 PubMed DOI

Callieri C. (2008). Picophytoplankton in freshwater ecosystems: the importance of small-sized phototrophs. Freshw. Rev. 1 1–28. 10.1608/FRJ-1.1.1 DOI

Callieri C., Coci M., Corno G., Macek M., Modenutti B., Balseiro E., et al. (2013). Phylogenetic diversity of nonmarine picocyanobacteria. FEMS Microbiol. Ecol. 85 293–301. 10.1111/1574-6941.12118 PubMed DOI

Callieri C., Cronberg G., Stockner J. G. (2012). ”Freshwater picocyanobacteria: single cells, microcolonies and colonial forms,” in Ecology of Cyanobacteria II: Their Diversity in Time and Space 2nd Edn ed. Whitton B. A. (Berlin: Springer; ) 229–269.

Callieri C., Stockner J. G. (2002). Freshwater autotrophic picoplankton: a review. J. Limnol. 61 1–14. 10.4081/jlimnol.2002.1 DOI

Camacho A. (2006). On the occurrence and ecological features of deep chlorophyll maxima (DCM) in Spanish stratified lakes. Limnetica 25 453–478.

Camacho A., Miracle M. R., Vicente E. (2003a). Which factors determine the abundance and distribution of picocyanobacteria in inland waters? A comparison among different types of lakes and ponds. Arch. Hydrobiol. 157 321–338. 10.1127/0003-9136/2003/0157-0321 DOI

Camacho A., Picazo A., Miracle M. R., Vicente E. (2003b). Spatial distribution and temporal dynamics of picocyanobacteria in a meromictic karstic lake. Algol. Stud. 109 171–184. 10.1127/1864-1318/2003/0109-0171 DOI

Camacho A., Vicente E., Miracle M. R. (2000). Spatio-temporal distribution and growth dynamics of phototrophic sulfur bacteria populations in the sulfide-rich Lake Arcas. Aquat. Sci. 62 334–349. 10.1007/PL00001339 DOI

Capella-Gutiérrez S., Silla-Martínez J. M., Gabaldón T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25 1972–1973. 10.1093/bioinformatics/btp348 PubMed DOI PMC

Cole J. R., Wang Q., Cardenas E., Fish J., Chai B., Farris R. J., et al. (2009). The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 37(suppl. 1) D141–D145. 10.1093/nar/gkn879 PubMed DOI PMC

Dufresne A., Ostrowski M., Scanlan D. J., Garczarek L., Mazard S., Palenik B. P., et al. (2008). Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria. Genome Biol. 9:R90 10.1186/gb-2008-9-5-r90 PubMed DOI PMC

Edgar R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32 1792–1797. 10.1093/nar/gkh340 PubMed DOI PMC

Edgar R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26 2460–2461. 10.1093/bioinformatics/btq461 PubMed DOI

Flombaum P., Gallegos J. L., Gordillo R. A., Rincón J., Zabala L. L., Jiao N., et al. (2013). Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus. Proc. Natl. Acad. Sci. U.S.A. 110 9824–9829. 10.1073/pnas.1307701110 PubMed DOI PMC

Frenkel A., Gaffron H., Battley E. H. (1950). Photosynthesis and photoreduction by the blue green alga, Synechococcus elongatus, Näg. Biol. Bull. 99 157–162. 10.2307/1538735 PubMed DOI

Fuller N. J., Marie D., Partensky F., Vaulot D., Post A. F., Scanlan D. J. (2003). Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea. Appl. Environ. Microbiol. 69 2430–2443. 10.1128/AEM.69.5.2430-2443.2003 PubMed DOI PMC

Ghai R., Hernandez C. M., Picazo A., Mizuno C. M., Ininbergs K., Díez B., et al. (2012). Metagenomes of Mediterranean coastal lagoons. Sci. Rep. 2:490 10.1038/srep00490 PubMed DOI PMC

Ghai R., Martin-Cuadrado A.-B., Molto A. G., Heredia I. G., Cabrera R., Martin J., et al. (2010). Metagenome of the Mediterranean deep chlorophyll maximum studied by direct and fosmid library 454 pyrosequencing. ISME J. 4 1154–1166. 10.1038/ismej.2010.44 PubMed DOI

Ghai R., Mizuno C. M., Picazo A., Camacho A., Rodriguez-Valera F. (2014). Key roles for freshwater Actinobacteria revealed by deep metagenomic sequencing. Mol. Ecol. 23 6073–6090. 10.1111/mec.12985 PubMed DOI

Guimarães P. I., Leão T. F., de Melo A. G. C., Ramos R. T. J., Silva A., Fiore M. F., et al. (2015). Draft genome sequence of the picocyanobacterium Synechococcus sp. strain GFB01 isolated from a freshwater lagoon in the Brazilian Amazon. Genome Announc. 3:e00876-15 10.1128/genomeA.00876-15 PubMed DOI PMC

Haft D. H., Loftus B. J., Richardson D. L., Yang F., Eisen J. A., Paulsen I. T., et al. (2001). TIGRFAMs: a protein family resource for the functional identification of proteins. Nucleic Acids Res. 29 41–43. 10.1093/nar/29.1.41 PubMed DOI PMC

Holtman C. K., Chen Y., Sandoval P., Gonzales A., Nalty M. S., Thomas T. L., et al. (2005). High-throughput functional analysis of the Synechococcus elongatus PCC 7942 genome. DNA Res. 12 103–115. 10.1093/dnares/12.2.103 PubMed DOI

Huang Y., Gilna P., Li W. (2009). Identification of ribosomal RNA genes in metagenomic fragments. Bioinformatics 25 1338–1340. 10.1093/bioinformatics/btp161 PubMed DOI PMC

Hyatt D., Chen G.-L., LoCascio P. F., Land M. L., Larimer F. W., Hauser L. J. (2010). Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119 10.1186/1471-2105-11-119 PubMed DOI PMC

Kashtan N., Roggensack S. E., Rodrigue S., Thompson J. W., Biller S. J., Coe A., et al. (2014). Single-cell genomics reveals hundreds of coexisting subpopulations in wild Prochlorococcus. Science 344 416–420. 10.1126/science.1248575 PubMed DOI

Kumar S., Stecher G., Tamura K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33 1870–1874. 10.1093/molbev/msw054 PubMed DOI PMC

Lassmann T., Sonnhammer E. L. (2005). Kalign–an accurate and fast multiple sequence alignment algorithm. BMC Bioinformatics 6:298 10.1186/1471-2105-6-298 PubMed DOI PMC

Laudenbach D. E., Grossman A. R. (1991). Characterization and mutagenesis of sulfur-regulated genes in a cyanobacterium: evidence for function in sulfate transport. J. Bacteriol. 173 2739–2750. 10.1128/jb.173.9.2739-2750.1991 PubMed DOI PMC

Lê S., Josse J., Husson F. (2008). FactoMineR: an R package for multivariate analysis. J. Stat. Softw. 25 1–18. 10.18637/jss.v025.i01 DOI

Li H., Durbin R. (2009). Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25 1754–1760. 10.1093/bioinformatics/btp324 PubMed DOI PMC

López-Pérez M., Ghai R., Leon M. J., Rodríguez-Olmos Á., Copa-Patiño J. L., Soliveri J., et al. (2013). Genomes of “Spiribacter”, a streamlined, successful halophilic bacterium. BMC Genomics 14:787 10.1186/1471-2164-14-787 PubMed DOI PMC

Lowe T. M., Eddy S. R. (1997). tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25 955–964. 10.1093/nar/25.5.0955 PubMed DOI PMC

MacIsaac E., Stockner J. G. (1993). “Enumeration of phototrophic picoplankton by autofluorescence microscopy,” in Handbook of Methods in Aquatic Microbial Ecology eds Sherr B., Sherr E. (Boca Raton, FL: CRC Press; ) 187–197.

Marie D., Partensky F., Jacquet S., Vaulot D. (1997). Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl. Environ. Microbiol. 63 186–193. PubMed PMC

Marin B., Nowack E. C., Glöckner G., Melkonian M. (2007). The ancestor of the Paulinella chromatophore obtained a carboxysomal operon by horizontal gene transfer from a Nitrococcus-like γ-proteobacterium. BMC Evol. Biol. 7:85 10.1186/1471-2148-7-85 PubMed DOI PMC

Martinez-Garcia M., Swan B. K., Poulton N. J., Gomez M. L., Masland D., Sieracki M. E., et al. (2012). High-throughput single-cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton. ISME J. 6 113–123. 10.1038/ismej.2011.84 PubMed DOI PMC

Mazard S., Ostrowski M., Partensky F., Scanlan D. J. (2012). Multi-locus sequence analysis, taxonomic resolution and biogeography of marine Synechococcus. Environ. Microbiol. 14 372–386. 10.1111/j.1462-2920.2011.02514.x PubMed DOI

Mongodin E. F., Nelson K., Daugherty S., Deboy R., Wister J., Khouri H., et al. (2005). The genome of Salinibacter ruber: convergence and gene exchange among hyperhalophilic bacteria and archaea. Proc. Natl. Acad. Sci. U.S.A. 102 18147–18152. 10.1073/pnas.0509073102 PubMed DOI PMC

Nawrocki E. (2009). Structural RNA Homology Search and Alignment using Covariance Models. St. Louis, MO: Washington University in St. Louis.

Nawrocki E. P., Eddy S. R. (2010). SSU-Align: A Tool for Structural Alignment of SSU rRNA Sequences. Available at: http://selab.janelia.org/software.html

Oh S., Caro-Quintero A., Tsementzi D., DeLeon-Rodriguez N., Luo C., Poretsky R., et al. (2011). Metagenomic insights into the evolution, function, and complexity of the planktonic microbial community of Lake Lanier, a temperate freshwater ecosystem. Appl. Environ. Microbiol. 77 6000–6011. 10.1128/AEM.00107-11 PubMed DOI PMC

Partensky F., Blanchot J., Vaulot D. (1999). Differential distribution and ecology of Prochlorococcus and Synechococcus in oceanic waters: a review. Bull. Inst. Oceanogr. Monaco Numero Spec. 19 457–476.

Peng Y., Leung H. C., Yiu S.-M., Chin F. Y. (2012). IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28 1420–1428. 10.1093/bioinformatics/bts174 PubMed DOI

Picazo A., Rochera C., Vicente E., Miracle M. R., Camacho A. (2013). Spectrophotometric methods for the determination of photosynthetic pigments in stratified lakes: a critical analysis based on comparisons with HPLC determinations in a model lake. Limnetica 32 139–158.

Porter K., Feig Y. (1980). The use of DAPI for identification and enumeration of bacteria and blue-green algae. Limnol. Oceanogr. 25 943–948. 10.4319/lo.1980.25.5.0943 DOI

Price G. D., Woodger F. J., Badger M. R., Howitt S. M., Tucker L. (2004). Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc. Natl. Acad. Sci. U.S.A. 101 18228–18233. 10.1073/pnas.0405211101 PubMed DOI PMC

Price M. N., Dehal P. S., Arkin A. P. (2010). FastTree 2–approximately maximum-likelihood trees for large alignments. PLoS ONE 5:e9490 10.1371/journal.pone.0009490 PubMed DOI PMC

Raes J., Korbel J. O., Lercher M. J., Von Mering C., Bork P. (2007). Prediction of effective genome size in metagenomic samples. Genome Biol. 8:R10 10.1186/gb-2007-8-1-r10 PubMed DOI PMC

Rice P., Longden I., Bleasby A. (2000). EMBOSS: the European molecular biology open software suite. Trends Genet. 16 276–277. 10.1016/S0168-9525(00)02024-2 PubMed DOI

Robertson B. R., Tezuka N., Watanabe M. M. (2001). Phylogenetic analyses of Synechococcus strains (cyanobacteria) using sequences of 16S rDNA and part of the phycocyanin operon reveal multiple evolutionary lines and reflect phycobilin content. Int. J. Syst. Evol. Microbiol. 51 861–871. 10.1099/00207713-51-3-861 PubMed DOI

Rocap G., Larimer F. W., Lamerdin J., Malfatti S., Chain P., Ahlgren N. A., et al. (2003). Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424 1042–1047. 10.1038/nature01947 PubMed DOI

Rodriguez-Valera F., Martin-Cuadrado A.-B., Rodriguez-Brito B., Pašiæ L., Thingstad T. F., Rohwer F., et al. (2009). Explaining microbial population genomics through phage predation. Nat. Rev. Microbiol. 7 828–836. 10.1038/nrmicro2235 PubMed DOI

Rozen S., Skaletsky H. (1999). Primer3 on the WWW for general users and for biologist programmers. Bioinform. Methods Protoc. 132 365–386.10.1385/1-59259-192-2:365 PubMed DOI

Sanchez-Baracaldo P., Handley B. A., Hayes P. K. (2008). Picocyanobacterial community structure of freshwater lakes and the Baltic Sea revealed by phylogenetic analyses and clade-specific quantitative PCR. Microbiology 154 3347–3357. 10.1099/mic.0.2008/019836-0 PubMed DOI

Sanchez-Baracaldo P., Hayes P., Blank C. (2005). Morphological and habitat evolution in the Cyanobacteria using a compartmentalization approach. Geobiology 3 145–165. 10.1111/j.1472-4669.2005.00050.x DOI

Scanlan D. J., Ostrowski M., Mazard S., Dufresne A., Garczarek L., Hess W. R., et al. (2009). Ecological genomics of marine picocyanobacteria. Microbiol. Mol. Biol. Rev. 73 249–299. 10.1128/MMBR.00035-08 PubMed DOI PMC

Shih P. M., Wu D., Latifi A., Axen S. D., Fewer D. P., Talla E., et al. (2013). Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc. Natl. Acad. Sci. U.S.A. 110 1053–1058. 10.1073/pnas.1217107110 PubMed DOI PMC

Six C., Thomas J.-C., Garczarek L., Ostrowski M., Dufresne A., Blot N., et al. (2007). Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study. Genome Biol. 8:R259. PubMed PMC

Steffen M. M., Li Z., Effler T. C., Hauser L. J., Boyer G. L., Wilhelm S. W. (2012). Comparative metagenomics of toxic freshwater cyanobacteria bloom communities on two continents. PLoS ONE 7:e44002 10.1371/journal.pone.0044002 PubMed DOI PMC

Sunagawa S., Coelho L. P., Chaffron S., Kultima J. R., Labadie K., Salazar G., et al. (2015). Structure and function of the global ocean microbiome. Science 348:1261359 10.1126/science.1261359 PubMed DOI

Tamura K., Nei M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10 512–526. PubMed

Tatusov R. L., Natale D. A., Garkavtsev I. V., Tatusova T. A., Shankavaram U. T., Rao B. S., et al. (2001). The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 29 22–28. 10.1093/nar/29.1.22 PubMed DOI PMC

Vadstein O. (2000). Heterotrophic, planktonic bacteria and cycling of phosphorus requirements, competitive ability and food web interactions. Adv. Microb. Ecol. 16 115–167. 10.1007/978-1-4615-4187-5_4 DOI

Veldhuis M. J., Kraay G. W. (2000). Application of flow cytometry in marine phytoplankton research: current applications and future perspectives. Sci. Mar. 64 121–134. 10.3989/scimar.2000.64n2121 DOI

Ventosa A., Nieto J. J., Oren A. (1998). Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62 504–544. PubMed PMC

West N. J., Schnhuber W. A., Fuller N. J., Amann R. I., Rippka R., Post A. F., et al. (2001). Closely related Prochlorococcus genotypes show remarkably different depth distributions in two oceanic regions as revealed by in situ hybridization using 16S rRNA-targeted oligonucleotides. Microbiology 147 1731–1744. 10.1099/00221287-147-7-1731 PubMed DOI

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