High-throughput single-cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton

ISME J. 2012 Jan;6(1):113-23. doi: 10.1038/ismej.2011.84. Epub 2011 Jun 30.


Recent discoveries suggest that photoheterotrophs (rhodopsin-containing bacteria (RBs) and aerobic anoxygenic phototrophs (AAPs)) and chemoautotrophs may be significant for marine and freshwater ecosystem productivity. However, their abundance and taxonomic identities remain largely unknown. We used a combination of single-cell and metagenomic DNA sequencing to study the predominant photoheterotrophs and chemoautotrophs inhabiting the euphotic zone of temperate, physicochemically diverse freshwater lakes. Multi-locus sequencing of 712 single amplified genomes, generated by fluorescence-activated cell sorting and whole genome multiple displacement amplification, showed that most of the cosmopolitan freshwater clusters contain photoheterotrophs. These comprised at least 10-23% of bacterioplankton, and RBs were the dominant fraction. Our data demonstrate that Actinobacteria, including clusters acI, Luna and acSTL, are the predominant freshwater RBs. We significantly broaden the known taxonomic range of freshwater RBs, to include Alpha-, Beta-, Gamma- and Deltaproteobacteria, Verrucomicrobia and Sphingobacteria. By sequencing single cells, we found evidence for inter-phyla horizontal gene transfer and recombination of rhodopsin genes and identified specific taxonomic groups involved in these evolutionary processes. Our data suggest that members of the ubiquitous betaproteobacteria Polynucleobacter spp. are the dominant AAPs in temperate freshwater lakes. Furthermore, the RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) gene was found in several single cells of Betaproteobacteria, Bacteroidetes and Gammaproteobacteria, suggesting that chemoautotrophs may be more prevalent among aerobic bacterioplankton than previously thought. This study demonstrates the power of single-cell DNA sequencing addressing previously unresolved questions about the metabolic potential and evolutionary histories of uncultured microorganisms, which dominate most natural environments.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actinobacteria / genetics
  • Actinobacteria / isolation & purification*
  • Actinobacteria / physiology
  • Bacteroidetes / genetics
  • Bacteroidetes / isolation & purification*
  • Bacteroidetes / physiology
  • Chemoautotrophic Growth
  • Flow Cytometry
  • Fresh Water / microbiology*
  • Metagenomics / methods
  • Molecular Sequence Data
  • Phototrophic Processes
  • Phylogeny
  • Proteobacteria / genetics
  • Proteobacteria / isolation & purification*
  • Proteobacteria / physiology
  • RNA, Ribosomal, 16S / genetics
  • Sequence Analysis, DNA / methods
  • Single-Cell Analysis / methods
  • Verrucomicrobia / isolation & purification*
  • Verrucomicrobia / physiology


  • RNA, Ribosomal, 16S