Linking Microbes to Their Genes at Single Cell Level with Direct-geneFISH

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Identifying specific microbial cells and their functional genes is crucial for understanding microbial communities.
• Current methods may not always link gene presence directly to individual cell identity.
• Fluorescence In Situ Hybridization (FISH) is a powerful technique for cellular identification.

Purpose of the Study:

• To introduce and describe the direct-geneFISH method.
• To enable direct linkage of gene presence to cell identity.
• To facilitate quantification of gene-carrying cells and gene copy numbers per cell.

Main Methods:

• Utilizes rRNA-targeting oligonucleotide probes for cell identification.
• Employs dsDNA polynucleotide probes with multiple fluorochromes for gene detection.
• Includes probe design, synthesis, the core protocol, microscopy, and data analysis.

Main Results:

• Directly links gene presence and potential metabolic capabilities to specific cell identities.
• Allows for the quantification of the cell fraction harboring a target gene.
• Enables determination of the number of target genes per cell.

Conclusions:

• Direct-geneFISH is a versatile method applicable to laboratory cultures and environmental samples.
• The protocol provides a comprehensive approach for gene-centric microbial cell analysis.
• This technique enhances our ability to study microbial functions at the single-cell level.