Revealing the in vivo growth and division patterns of mouse gut bacteria

Abstract

Current techniques for studying gut microbiota are unable to answer some important microbiology questions, like how different bacteria grow and divide in the gut. We propose a method that integrates the use of sequential d-amino acid-based in vivo metabolic labeling with fluorescence in situ hybridization (FISH), for characterizing the growth and division patterns of gut bacteria. After sequentially administering two d-amino acid-based probes containing different fluorophores to mice by gavage, the resulting dual-labeled peptidoglycans provide temporal information on cell wall synthesis of gut bacteria. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, including species that cannot be cultured separately in vitro, are revealed. Our method offers a facile yet powerful tool for investigating the in vivo growth dynamics of the bacterial gut microbiota, which will advance our understanding of bacterial cytology and facilitate elucidation of the basic microbiology of this gut "dark matter."

Fig. 1. Schematic illustration of the FDAA-based labeling strategy integrated with FISH staining and the two-color fluorescence imaging of the sequentially labeled gut microbiota.

(A) TADA and Cy5ADA were given to mouse by gavage at an interval of 3 hours. The cecal microbiota was collected and imaged, and the taxonomic identifications of different bacteria were then determined by corresponding FISH probes. (B) Two-color fluorescence imaging of the gut bacteria sequentially labeled by TADA (green) and Cy5ADA (red). Scale bar, 10 μm. Representative images from at least three independent experiments are shown. BF, Bright field. (C) Zoomed in view of the indicated bacteria from the merged image above. The green and red colors revealed the distinct growth patterns of different bacteria. Scale bars, 2 μm.

Fig. 2. Classical bacterial growth and division patterns could be observed in the labeled gut microbiota.

Following STAMP (TADA and then Cy5ADA gavage), the mouse cecal microbiota was collected and analyzed by confocal fluorescence microscopy. In the cartoon, gray regions represent the sites of active cell wall synthesis, and green and red indicates the newly constructed PGN. (A) Diffuse synthesis of PGN. (B) Spiral synthesis of PGN. (C) Septum synthesis dominating the cell division. (D) Polar growth. (E) Stalk/budding formation. Scale bars, 2 μm.

Fig. 3. Confocal fluorescence imaging of 12 FDAA-labeled and FISH-stained gut bacterial genera.

The cecal microbiotas of mice received sequential labeling of TADA (green) and Cy5ADA (red) were stained by different FISH probes (blue) targeting corresponding genera. (A to F) Representative images of FDAA-labeled bacteria in six Gram-negative genera. Scale bars, 1 μm. (G to L) Representative images of FDAA-labeled bacteria in six Gram-positive genera. Scale bars, 5 μm. Photographs of bacteria, representing consistent labeling pattern in each genus from at least three independent FISH experiments, are presented.

Fig. 4. Clostridium shows diverse cellular growth and division patterns.

(A) Confocal fluorescence imaging of the polyphenotypic bacteria in the Clostridium genus identified by FISH staining. Scale bar, 10 μm. (B) Three FISH probes targeting corresponding Clostridium species showed their distinct FDAA labeling patterns. Scale bars, 2 μm. Photographs of bacteria, representing consistent labeling pattern in each species from at least three independent FISH experiments, are presented. ATCC, American Type Culture Collection.

Fig. 5. Confocal fluorescence imaging of FDAA-labeled and FISH-stained gut bacterial species.

The growth patterns of three species that are culturable in vitro (A to C) and three species that have not been cultured separately in the laboratory (D to F) are revealed. Scale bars, 2 µm. Bacterial micrographs demonstrate consistent labeling patterns in each species from at least three independent FISH experiments.

Fig. 6. Confocal fluorescence imaging of the sequentially labeled SFB.

Some characteristic elements of SFB, including segments at varied differentiation stages (A), intrasegmental bodies (B), needle-like holdfast and triseptum in an asymmetric division location (C), and a symmetric division locus (D), were readily observed in the FDAA-labeled bacteria. Scale bar, 10 μm.