Strain-level profiling with picodroplet microfluidic cultivation reveals host-specific adaption of honeybee gut symbionts

Abstract

Background Symbiotic gut microbes have a rich genomic and metabolic pool and are closely related to hosts’ health. Traditional sequencing profiling masks the genomic and phenotypic diversity among strains from the same species. Innovative droplet-based microfluidic cultivation may help to elucidate the inter-strain interactions. A limited number of bacterial phylotypes colonize the honeybee gut, while individual strains possess unique genomic potential and critical capabilities, which provides a particularly good model for strain-level analyses. Results Here, we construct a droplet-based microfluidic platform and generated ~ 6 × 108 droplets encapsulated with individual bacterial cells from the honeybee gut and cultivate in different media. Shotgun metagenomic analysis reveals significant changes in community structure after droplet-based cultivation, with certain species showing higher strain-level diversity than in gut samples. We obtain metagenome-assembled genomes, and comparative analysis reveal a potential novel cluster from Bifidobacterium in the honeybee. Interestingly, Lactobacillus panisapium strains obtained via droplet cultivation from Apis mellifera contain a unique set of genes encoding l-arabinofuranosidase, which is likely important for the survival of bacteria in competitive environments. Conclusions By encapsulating single bacteria cells inside microfluidic droplets, we exclude potential interspecific competition for the enrichment of rare strains by shotgun sequencing at high resolution. The comparative genomic analysis reveals underlying mechanisms for host-specific adaptations, providing intriguing insights into microbe-microbe interactions. The current approach may facilitate the hunting for elusive bacteria and paves the way for large-scale studies of more complex animal microbial communities.

Potential Source and Transmission Pathway of Gut Bacteria in the Diamondback Moth, Plutella xylostella

“…This study found that many bacteria in food can be transferred to the gut of P. xylostella by the traditional culture method, but this method is based on the similarity of the 16S rDNA of isolated bacteria. Although the similarity of most bacteria is more than 99%, based on the current study, the bacterial taxonomic units have entered the level of strains, and these bacteria with highly similar 16S rDNA belong to the same genus; however, they may belong to different strains [ 34 ]. Therefore, it is necessary to study the correlation between gut bacteria of P. xylostella and food more accurately and systematically by bacterial markers and other methods in the future.…”

Identification of the mutual gliding locus as a factor for gut colonization in non-native bee hosts using the ARTP mutagenesis

“…After colonization, the bees were given sterilized pollen and sugar water (50%, w/v ) ad libitum for 10 days. On day 5 and day 10, 6 bees were randomly selected from each cup cage, and the entire guts were aseptically dissected by gently pulling the stingers without touching the abdomen surface using sterilized forceps [ 23 ]. Subsequently, the dissected guts were crushed in 100 μL 25% ( v/v ) glycerol using an electric tissue grinder (OSE-Y30; Tiangen Biotech, Beijing, China).…”

“…Although they share core gut microbiota mainly composed of five phylotypes: Gilliamella , Snodgrassella , Bifidobacterium , Lactobacillus Firm4, and Firm5, phylogenetic analysis shows that strains from different bee species separate into divergent lineages [ 19 , 20 ]. Previous genomic analyses show that some strains from Gilliamella , Lactobacillus Firm5, and Bifidobacterium exhibit different metabolic potentials for carbohydrates, which may be relevant to the adaption of bee hosts [ 21 – 23 ]. Interestingly, Snodgrassella strain wkB12 from Bombus bimaculatus could not colonize the A. mellifera gut, while cross-host microbe transfer is observed across Apis species [ 18 ].…”

Strategies to increase the robustness of microbial cell factories

“…Second, ALE is an efficient tool for engineering microbial cells with specific phenotypes, whereas the isolation of the target mutant from a microflora usually requies a high-throughput facility. For example, the DREM CELL platform allows for the screening of target strains at a picoliter scale (Meng et al 2022). Depending on the fluorescence output, a biosensor based on transcription factors or riboswitches can significantly increase the efficiency of a screening process (Li et al 2023).…”