Visualizing the Growth and Division of Rat Gut Bacteria by D-Amino Acid-Based in vivo Labeling and FISH Staining

Abstract

Rat is a widely used mammalian model for gut microbiota research. However, due to the difficulties of individual in vitro culture of many of the gut bacteria, much information about the microbial behaviors in the rat gut remains largely unknown. Here, to characterize the in situ growth and division of rat gut bacteria, we apply a chemical strategy that integrates the use of sequential tagging with D-amino acid-based metabolic probes (STAMP) with fluorescence in situ hybridization (FISH) to rat gut microbiota. Following sequential gavages of two different fluorescent D-amino acid probes to rats, the resulting dually labeled gut bacteria provides chronological information of their in situ cell wall synthesis. After taxonomical labeling with FISH probes, most of which are newly designed in this study, we successfully identify the growth patterns of 15 bacterial species, including two that have not been cultured separately in the laboratory. Furthermore, using our labeling protocol, we record Butyrivibrio fibrisolvens cells growing at different growth stages of a complete cell division cycle, which offers a new scope for understanding basic microbial activities in the gut of mammalian hosts.

Metabolic fluorine labeling and hotspot imaging of dynamic gut microbiota in mice

“…As expected, Gram-positive bacteria showed more efficient labeling than Gram-negative bacteria. This is consistent with previous fluorescent DAA labeling results (36,37) and can be explained by the fact that the PGN layers of Gram-positive bacteria are much thicker than those of Gram-negative bacteria. Further quantitative analysis of the results revealed that the number of labeled fluorine was ~10 8 atoms per bacterium (see table S2 for calculation).…”

Development and Applications of D‐Amino Acid Derivatives‐based Metabolic Labeling of Bacterial Peptidoglycan

“…When combined with the use of FISH, a classical method to determine bacterial taxonomic information, the bacterial growth patterns at the genus and species level were identified. In their subsequent work, the above method was applied to analyze the growth and division patterns of human gut bacteria by colonizing germ‐free mice with human fecal microbiota and the growth and division patterns of rat gut bacteria [41f,42] …”