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.”

Imaging the in vivo growth patterns of bacteria in human gut Microbiota

“…Different distributions of the two-color fluorescence among various bacteria provided a chronological recording of the PGN synthesis in each bacterium, and stronger labeling of the second FDAA probe (Cy5ADA, shown in red) represented sites with more active PGN constructions. 5 Most bacteria exhibited zonal growth, where the addition of new PGN was concentrated to a specific area, such as elongating near the division plane ( Figure 1(c ), No. 1), growing at one pole of the cell ( Figure 1(c ), No.…”

“…The FISH probes used were either previously used in literatures or designed de novo using an algorithm previously reported. 5 , 12 In this study, we further optimized the algorithm to prevent the formation of secondary DNA structures and improve the hybridization success rates of the designed sequences. The specificities of the designed FISH probes targeting each species were carefully verified from multiple aspects: 1) consistent labeling patterns and morphogenesis of the bacteria stained by the same probe (Figure S3); 2) if possible, agreeing results between the labeling ratio of the FISH-stained bacteria by flow cytometry and the corresponding relative abundance determined by metagenomic sequencing (Figure S4); 3) when some species only existed in either the donor 1 or the donor 2 group, the two groups of human-derived microbiotas were used as the control group for each other when testing FISH probes of these species.…”

“… 3 , 4 Recently, we reported a strategy that could reveal the indigenous growth and division patterns of different bacterial genus or species in the gut microbiota of mice and rats. 5 , 6 This integrative method combined the use of in vivo sequential tagging with fluorescent D-amino acid-based probes (STAMP) and fluorescent in situ hybridization (FISH). 5 Fluorescent D-amino acid (FDAA) probes can metabolically label peptidoglycan (PGN) by the functioning of bacterial D,D and L,D-transpeptidases, which play pivotal roles in their PGN constructions.…”

“… 5 , 6 This integrative method combined the use of in vivo sequential tagging with fluorescent D-amino acid-based probes (STAMP) and fluorescent in situ hybridization (FISH). 5 Fluorescent D-amino acid (FDAA) probes can metabolically label peptidoglycan (PGN) by the functioning of bacterial D,D and L,D-transpeptidases, which play pivotal roles in their PGN constructions. 7 , 8 The sequential labeling with gavaged FDAAs containing different fluorophores chronologically recorded the morphogenesis of most bacteria in the gut, including those that had not been cultured individually in vitro .…”

Spatial profiling of microbial communities by sequential FISH with error-robust encoding

“…SEER-FISH provides an unprecedented method for profiling the spatial ecology of complex microbial communities in situ. the environment and hosts [8][9][10][11] .Fluorescence in situ hybridization (FISH) with probes targeting ribosomal RNA (rRNA) has been widely used to identify specific microbial taxa and allows for in situ spatial analysis of microbiomes at single cell resolution [12][13][14][15][16][17][18] . One challenge of this spatial profiling is the huge phylogenetic and functional diversity of free-living and host-associated microbial communities.…”