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

[Abstract] 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. Pseudomonas aeruginosa 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 cdrA :: gfp ) 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 ( pelA , pslA ). This protocol can be adapted by others to isolate subpopulations of high- and low- c-di-GMP P. aeruginosa 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.

Phenotypic heterogeneity is a common phenomenon in nature, occurring when a population of genetically identical organisms (in the present case, bacteria) display diverse behaviors, despite experiencing the same environmental conditions (Ackermann, 2015). Testing hypotheses related to changes in gene expression in phenotypically heterogeneous populations requires the ability to separate out these sub-populations. For example, when analyzed as a single batch (combining both subpopulations together), the high gene expression by one sub-population would be averaged with the low expression from the other sub-population, resulting in the erroneous conclusion that expression of the gene of interest is at some intermediate level. Therefore, the purpose of developing this protocol was to separate genetically identical, but apparently physiologically distinct sub-populations of P. aeruginosa cells with high and low intracellular levels of c-di-GMP in order to determine whether these two subpopulations may differ in their expression of genes involved in biofilm formation (genes encoding Pel and Psl polysaccharide biosynthetic machineries). The protocol uses either of two versions of a plasmidbased, transcriptional reporter of c-di-GMP levels in P. aeruginosa (pPcdrA::gfp and pPcdrA::gfpASV) (Armbruster et al., 2019). These two reporter plasmids differ by which GFP allele they express, either the stable GFP (GFPmut3) or an unstable variant with a shorter half-life (GFPASV) (Andersen et al., 1998;Rybtke et al., 2012). The benefit of the stable GFP version of the reporter is that it tends to have a higher dynamic range, whereas the benefit of the short half-life GFP version is that it can be used in time course experiments when the levels of c-di-GMP may decrease. The protocol also makes use of a number of controls to help draw flow cytometry gates that separate cells with high and low c-di-GMP reporter 3 www.bio-protocol.org/e3891  activity (as measured by GFP fluorescence). Controls for gating include promotorless vector control plasmids harboring stable or unstable GFP (pMH487 or pMH489) and 2 P. aeruginosa strains with clean deletions of key proteins in the Wsp chemosensory system (PAO1 ∆wspR and PAO1 ∆wspF ∆pelC ∆pslBCD) that will have constitutively low or high levels of c-di-GMP reporter activity (Güvener and Harwood, 2007). is attached to a surface, c-di-GMP binds to FleQ, the cdrA promoter is de-repressed, and GFP is made. Yellow star = c-di-GMP. RBSII is an enhanced ribosomal binding site, T0 and T1 are transcriptional terminators, and Gent R and Amp R represent resistance genes for gentamicin and ampicillin, respectively. This is the Seattle c-di-GMP reporter that was originally published in Rybtke The Wsp system is a "surface sensing" system in P. aeruginosa with homology to bacterial chemosensory signal transduction complexes, that produces c-di-GMP in response to surface contact (Hickman et al., 2005). The Wsp system senses a yet to be identified signal related to surface contact through WspA, a membrane-bound receptor homologous to methyl-accepting chemotaxis proteins ∆wspR deletion strain is incapable of producing c-di-GMP through the Wsp system and is a helpful control strain for gating a "low c-di-GMP" population. In contrast, the PAO1 ∆wspF ∆pelC ∆pslBCD strain has constitutively high c-di-GMP produced through the Wsp system due to the deletion of the wspF methylesterase gene. This strain also has deletions in genes required for Pel and Psl polysaccharide biosynthesis (∆pelC ∆pslBCD), to abrogate clumping due to polysaccharide overproduction in the PAO1 ∆wspF background.

Data analysis
1. Experiments should be performed in biological triplicates. For this protocol, a biological replicate is defined as a biofilm grown, harvested, and flow-sorted on a separate experimental day.
2. We used the comparative Ct method (ΔΔCt) (Schmittgen and Livak, 2008) to determine the fold change in target gene expression between the reporter "on" and "off" populations.
a. From the qRT-PCR dataset, calculate the ∆Ct for each experimental gene (e.g., pelA, pslA) in either the sorted "on" or "off" populations by subtracting the Ct of the control gene (ampR) from the experimental gene's Ct. For example, this is the ∆Ct for pelA in biofilm cells that were sorted as having high c-di-GMP reporter activity: Then, calculate the ΔΔCt by subtracting the ΔCt of pelA in the reporter "off" population from the ΔCt of pelA in the reporter "on" population.
c. Finally, to determine the fold change in expression in the reporter "on" population compared to the reporter "off" population, take 2^-ΔΔCt. Data can now be presented as the average fold change in PelA or PslA expression in the PcdrA reporter sorted "on" population (high GFP) 12 www.bio-protocol.org/e3891 relative to the "off" population (low GFP) for the three biological replicates (see Figure 4 for an example of how these data can be presented). Combine the ingredients to a final volume of 950 ml in purified and deionized water, adjust pH to 7.4, then add more water until the final volume is 1 L.