Analysis of c-di-GMP High and Low Subpopulations Using Flow-assisted Cell Sorting (FACS) and Quantitative Reverse Transcriptase PCR (qRT-PCR).

Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently used a plasmid-based c-di-GMP reporter (pP ) to describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells during early biofilm formation. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. Here, we describe the protocol for a key experiment to confirm our initial observation of c-di-GMP heterogeneity during surface sensing: the use of flow-assisted cell sorting (FACS) to isolate subpopulations of cells with high and low c-di-GMP reporter activity, followed by quantitative Reverse Transcriptase PCR (qRT-PCR) of genes that are known to be transcriptionally regulated in response to cellular c-di-GMP levels (). This protocol can be adapted by others to isolate subpopulations of high- and low- c-di-GMP cells that are genetically identical, but phenotypically distinct for future experiments examining specific mRNA transcripts as we did or, presumably, for additional applications like RNAseq, proteomics, or TNseq. .