Stable, fluorescent markers for tracking synthetic communities and assembly dynamics

Area of Science:

• Microbiology
• Synthetic Biology
• Systems Biology

Background:

• Microbiome research has advanced from description to mechanistic understanding of microbial community assembly.
• Synthetic communities (SynComs) are crucial for studying assembly dynamics, but require precise quantification.
• Existing methods struggle with absolute quantification and differentiating multiple members within a SynCom.

Purpose of the Study:

• To develop a novel method for absolute quantification and differentiation of microbes in SynComs.
• To enable tracking of microbial assembly dynamics in complex environments.
• To overcome limitations of current sequencing- and culturing-based approaches.

Main Methods:

• Developed differential fluorescent marking (DFM) using three distinguishable fluorescent proteins.
• Utilized the mini-Tn7 transposon system for stable integration in Proteobacteria.
• Constructed DFM using the pTn7-SCOUT plasmid family for modular assembly.
• Employed flow cytometry for differentiating, quantifying, and tracking SynCom members.

Main Results:

• DFM strategy demonstrated stability and broad applicability across Proteobacteria.
• DFM showed no detrimental effects on microbial fitness or community assembly.
• Successfully differentiated, quantified, and tracked a six-member SynCom.
• Observed distinct colonization dynamics in pea and barley root rhizospheres.

Conclusions:

• DFM provides a powerful, culture- and sequencing-independent tool for microbiome assembly studies.
• This method facilitates mechanistic insights into microbial community dynamics.
• Opens new research avenues for understanding complex microbial ecosystems.