Single-Cell Resolution of Uncultured Magnetotactic Bacteria via Fluorescence-Coupled Electron Microscopy

• Published in: Applied and environmental microbiology 2017-06, Vol.83 (12)
• Main Authors: Li, Jinhua, Zhang, Heng, Menguy, Nicolas, Benzerara, Karim, Wang, Fuxian, Lin, Xiaoting, Chen, Zhibao, Pan, Yongxin
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Typing Techniques - instrumentation; Bacterial Typing Techniques - methods; Cells; China; Crystals; Electron microscopy; Elongation; Estuaries; Estuarine environments; Fluorescence; Fluorescence in situ hybridization; Fluvial sediments; Functional groups; Gammaproteobacteria - classification; Gammaproteobacteria - genetics; Gammaproteobacteria - isolation & purification; Gammaproteobacteria - ultrastructure; Gene sequencing; Genome, Bacterial; Geologic Sediments - microbiology; In Situ Hybridization, Fluorescence; Lake sediments; Lakes - microbiology; Magnetism; Magnetite; Magnetosomes - chemistry; Magnetosomes - genetics; Magnetosomes - ultrastructure; Methods; Microorganisms; Microscopy, Electron; Mineralization; Phylogeny; Prisms; Probes; Rivers; Rivers - microbiology; rRNA 16S; Scanning electron microscopy; Sediments; Single-Cell Analysis - instrumentation; Single-Cell Analysis - methods; Strategy
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.00409-17

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) fluorescence hybridization analyses coordinated with (iv) transmission or scanning electron microscopy of the probe-hybridized cells. The application of this strategy identified a magnetotactic strain, 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 fluorescence hybridization (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. Magnetotactic 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 magnetotactic strain,