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Article

Diverse uncultivated ultra-small bacterial cells in groundwater

  • Nature Communications 6, Article number: 6372 (2015)
  • doi:10.1038/ncomms7372
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Abstract

Bacteria from phyla lacking cultivated representatives are widespread in natural systems and some have very small genomes. Here we test the hypothesis that these cells are small and thus might be enriched by filtration for coupled genomic and ultrastructural characterization. Metagenomic analysis of groundwater that passed through a ~0.2-μm filter reveals a wide diversity of bacteria from the WWE3, OP11 and OD1 candidate phyla. Cryogenic transmission electron microscopy demonstrates that, despite morphological variation, cells consistently have small cell size (0.009±0.002 μm3). Ultrastructural features potentially related to cell and genome size minimization include tightly packed spirals inferred to be DNA, few densely packed ribosomes and a variety of pili-like structures that might enable inter-organism interactions that compensate for biosynthetic capacities inferred to be missing from genomic data. The results suggest that extremely small cell size is associated with these relatively common, yet little known organisms.

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Acknowledgements

Support for this research was provided by the U.S. Department of Energy under contracts DE-SC0004733 and ER65009 (Subsurface Biogeochemistry Program, Biological and Environmental Research), and DE-AC02-05CH11231 (Office of Basic Energy Sciences, Berkeley Synchrotron Infrared Structural Biology Program, and DOE Joint Genome Institute). We thank R. Csencsits for help with microscopy, S.W. Mullin for assistance with clone library construction, L.A. Hug for input to phylogenetic analyses and I. Zweimüller for statistics support.

Author information

Author notes

    • Birgit Luef

    Present addresses: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim 7491, Norway

    • Kyle R. Frischkorn

    Present addresses: Department of Earth and Environmental Science, Columbia University, Palisades, New York 10964-800, USA

    • Kelly C. Wrighton

    Present addresses: Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA

    • Luis R. Comolli

    Present addresses: ALS-Molecular Biology Consortium, Advanced Light Source Beamline 4.2.2, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

Affiliations

  1. Department of Earth and Planetary Science, University of California, Berkeley, California 94720, USA

    • Birgit Luef
    • , Kyle R. Frischkorn
    • , Kelly C. Wrighton
    • , Brian C. Thomas
    • , Andrea Singh
    • , Cristina E. Siegerist
    •  & Jillian F. Banfield
  2. Ecology Department, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Hoi-Ying N. Holman
    •  & Giovanni Birarda
  3. Geophysics Department Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Kenneth H. Williams
    •  & Jillian F. Banfield
  4. Department of Energy (DOE), Joint Genome Institute, Walnut Creek, California 94598, USA

    • Susannah G. Tringe
  5. Structural Biology and Imaging Department, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Kenneth H. Downing
    •  & Luis R. Comolli
  6. Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720, USA

    • Jillian F. Banfield

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Contributions

B.L. contributed to the study design, collected, concentrated and cryo-plunged samples, performed CARD-FISH, cryo-TEM surveys, collected and reconstructed electron tomograms, conducted data analysis and wrote the paper. K.R.F. extracted DNA, constructed and analysed the clone library, genetically engineered the E. coli probe control, carried out some phylogenetic analysis and designed CARD-FISH probes. K.C.W. contributed to the study design and sampling, and performed EMIRGE analysis. H.-Y.N.H. and G.B. performed SIR spectromicroscopy. B.C.T. and A.S. contributed to the bioinformatics analyses. K.H.W. conducted the field experiment. C.E.S. wrote the program for cropping subtomographic volumes with orientation tracking, computed the S-layer 3D reconstructions and selected subtomographic volumes. S.G.T. oversaw metagenomic sequencing. K.H.D. provided the cryo-TEM infrastructure. L.R.C. assisted with the cryo-TEM surveys, collection of tomographic data sets, acquisition of tomographic data for subtomographic averaging, tomographic reconstructions, processing and analysis and conducted the S-layer 3D reconstruction by subtomographic averaging. J.F.B. contributed to the study design, analysed metagenomic and other data and co-wrote the paper. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Luis R. Comolli or Jillian F. Banfield.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1-14, Supplementary Tables 1-8, Supplementary Notes 1-6 and Supplementary References

Videos

  1. 1.

    Supplementary Movie 1

    Tomographic reconstruction of an ultra-small cell. Note, the very dense cytoplasmic compartment and conspicuous, complex cell wall enveloped by a periodic S-layer. Note, that as the movie slices through the cell, the spiral structure is particularly evident. The volume of the spiral is consistent its identification as tightly packed genomic DNA. The high contrast sub-cellular bodies located at both poles of the cell are inferred to be ribosomes.

  2. 2.

    Supplementary Movie 2

    3D reconstruction of an ultra-small cell. This cell surface has filamentous appendages of multiple types: one longer, thicker pilus-like structure (left bottom side of the bacterium), and shorter pili-like structures sparsely distributed across the cell surface.

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