In situ identifying sennoside A-reducing bacteria guilds in human gut microbiota via enzymatic activity visualization

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Microbial functions in the gut are complex and not fully predictable from genomics alone.
• Intracellular enzyme activity often doesn't directly correlate with a bacterium's overall function.
• Existing methods struggle to link enzymatic activity to specific microbial strains in situ.

Purpose of the Study:

• To develop a platform for visualizing and isolating bacteria based on active intracellular enzyme activity.
• To link enzymatic function to localization, identification, and isolation of microbial strains.
• To identify functional guilds and novel enzymes within complex microbial communities.

Main Methods:

• Developed an enzymatic activity visualization platform using substrate-based probes targeting isozymes.
• Utilized alkyne-azide cycloaddition for fluorescent visualization of enzyme activity in anaerobic gut microbiota.
• Integrated platform with fluorescence-activated cell sorting and 16S rRNA sequencing for isolation and identification.
• Validated probe selectivity using metabolic tests, fluorescence imaging, and proteomics on reference strains.

Main Results:

• Successfully visualized and validated bacterial cell-level enzyme activity and probe selectivity.
• Identified functional guilds responsible for sennoside A reduction in the gut microbiota.
• Discovered that phylogenetically diverse bacteria can perform similar metabolic functions.
• Identified a novel sennoside A-reducing enzyme, StNfrA, and confirmed its activity.

Conclusions:

• The enzymatic activity visualization platform reliably localizes, isolates, and identifies functional microbial guilds.
• The platform enables discovery of novel enzymes and understanding of metabolic functions in complex microbiomes.
• This approach advances the study of microbial biochemistry and function in their native environments.