title: Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy.
abstract: Magnetotactic bacteria (MTB) form intracellular chain-assembled nanocrystals of magnetite or greigite termed magnetosomes. The characterization of magnetosome crystals requires electron microscopy due to their nanoscopic sizes. However, electron microscopy does not provide phylogenetic information for MTB. We have developed a strategy for the simultaneous and rapid phylogenetic and biomineralogical characterization of uncultured MTB at the single-cell level. It consists of four steps: (i) enrichment of MTB cells from an environmental sample, (ii) 16S rRNA gene sequencing of MTB, and (iii) fluorescencein situhybridization analyses coordinated with (iv) transmission or scanning electron microscopy of the probe-hybridized cells. The application of this strategy identified a magnetotacticGammaproteobacteriastrain, SHHR-1, from brackish sediments collected from the Shihe River estuary in Qinhuangdao City, China. SHHR-1 magnetosomes are elongated prismatic magnetites which can be idealized as hexagonal prisms. Taxonomic groups of uncultured MTB were also identified in freshwater sediments from Lake Miyun in northern Beijing via this novel coordinated fluorescence and scanning electron microscopy method based on four group-specific rRNA-targeted probes. Our analyses revealed that major magnetotactic taxonomic groups can be accurately determined only with coordinated scanning electron microscopy observations on fluorescently labeled single cells due to limited group coverage and specificity for existing group-specific MTB fluorescencein situhybridization (FISH) probes. Our reported strategy is simple and efficient, offers great promise toward investigating the diversity and biomineralization of MTB, and may also be applied to other functional groups of microorganisms.IMPORTANCEMagnetotactic bacteria (MTB) are phylogenetically diverse and biomineralize morphologically diverse magnetic nanocrystals of magnetite or greigite in intracellular structures termed magnetosomes. However, many uncultured MTB strains have not been phylogenetically identified or structurally investigated at the single-cell level, which limits our comprehensive understanding of the diversity of MTB and their role in biomineralization. We developed a fluorescence-coupled electron microscopy method for the rapid phylogenetic and biomineralogical characterization of uncultured MTB at the single-cell level. Using this novel method, we successfully identified taxonomic groups of several uncultured MTB and one novel magnetotacticGammaproteobacteriastrain, SHHR-1, from natural environments. Our analyses further indicate that strain SHHR-1 forms elongated prismatic magnetites. Our findings provide a promising strategy for the rapid characterization of phylogenetic and biomineralogical properties of uncultured MTB at the single-cell level. Furthermore, due to its simplicity and generalized methodology, this strategy can also be useful in the study of the diversity and biomineralization properties of microbial taxa involved in other mineralization processes.
authors.affiliation: Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, People's Republic of China lijinhua@mail.iggcas.ac.cn.|Laboratory for Marine Geology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, People's Republic of China.|France-China Biomineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.|Laboratory for Marine Geology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, People's Republic of China.|France-China Biomineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing, People's Republic of China.|College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, People's Republic of China.
authors.forename: Heng
authors.initials: H
authors.lastname: Zhang
authors.affiliation: France-China Biomineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing, People's Republic of China.|IMPMC, CNRS UMR 7590, Sorbonne Universités, MNHN, UPMC, IRD UMR 206, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Menguy
authors.affiliation: IMPMC, CNRS UMR 7590, Sorbonne Universités, MNHN, UPMC, IRD UMR 206, Paris, France.
authors.forename: Karim
authors.initials: K
authors.lastname: Benzerara
authors.affiliation: Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.|Laboratory for Marine Geology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, People's Republic of China.|France-China Biomineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Fuxian
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.|College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, People's Republic of China.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Lin
authors.affiliation: College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, People's Republic of China.
authors.forename: Zhibao
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.|France-China Biomineralization and Nano-Structures Laboratory, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Pan
mesh_terms: D015373:Bacterial Typing Techniques / Q000295:instrumentation / Q000379:methods*; D002681:China; D020563:Gammaproteobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D016680:Genome, Bacterial; D019015:Geologic Sediments / Q000382:microbiology*; D017404:In Situ Hybridization, Fluorescence; D060106:Lakes / Q000382:microbiology*; D055944:Magnetosomes / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D008854:Microscopy, Electron; D010802:Phylogeny; D045483:Rivers / Q000382:microbiology*; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
keywords: FISH; TEM; biomineralization; fluorescence-coupled electron microscopy; magnetotactic bacteria