Development of the fluorescent imaging method of single-molecule sRNA SgrS <italic>in situ</italic> in <italic>Escherichia coli</italic>

Abstract

Single-cell gene expression studies could not only provide the real-time information on intracellular amount and subcellular localization of RNA, but also could reveal the regulatory mechanism of bacterial variation, which cannot be obtained based on traditional view of bacterial populations. Owing to the application of green fluorescent protein (GFP) reporters, most of studies have mainly focused on proteins. Recently, the quantitative and localization of bacterial RNAs have been also implemented at single transcript level. All of the non-coding RNAs who function as crucial regulators of gene expression are called regulatory small RNAs (sRNA) in bacteria. Single molecular imaging techniques for labeling bacterial sRNA can be used to detect the activity and abundance of sRNA, which will help us to understand its regulatory mechanism and biological function. SgrS, a conservative sRNA in γ-proteobacteria, is highly expressed under glucose-phosphate stress and involve in the sugar metabolism in bacteria. We chose SgrS as a model to establish the single-molecule fluorescence in situ hybridization (smFISH) method of RNA detection at the single-molecule level in Escherichia coli . It will be helpful for further studies on sRNA subcellular localization and gene regulation. We used kanamycin resistance box replace sgrS to successful construct sgrS deletion mutant Δ sgrS and subsequently cured the helper plasmid pKD46. The SgrS overexpression strain Δ sgrS -pBAD-SgrS was generated by introducing SgrS-overexpressed plasmid into the Δ sgrS strains. E. coli MG1655, Δ sgrS and Δ sgrS -pBAD-SgrS, all E. coli K-12 derivatives, were used in this study. The smFISH method on SgrS in E. coli was established. The optimal conditions are briefly stated as follows. The cells were fixed in 3.7% formaldehyde at room temperature for 30 minutes. Cells were permeabilized at 4°C for overnight. Washed cells with 40% wash solution was resuspended in 40% hybridization buffer. The cells were centrifuged and resuspended in the hybridization buffer with 1 μM sRNAs probes that denatured at 98°C for 10 minutes. Hybridization reactions were incubated in the hybridization oven at 40°C for 3 hours. The cells were washed five times with 40% wash solution. During wash, cells were resuspended and incubated at 40°C for 30 minutes. Cells were pelleted by 600× g centrifugation for 3 minutes, and the supernatant was removed. Cells were resuspended with 100 μL of freshly prepared 5 μg mL− 1 Alexa-555 WGA in 1×PBS, and incubated at room temperature for 10 minutes. Cells were pelleted by 600× g centrifugation for 3 minutes, and the supernatant was removed. The cells were resuspended in 5 μL of 2×SSC. Then 1 μL of sample and 1 μL of SlowFade Diamond Antifade Mountant were pipetted to poly-L-lysine treated borosilicate chambered coverslip. A coverslip was placed on the sample. Super-resolution imaging was performed on Nikon inverted microscope with a 100× oil immersion objective. SgrS was indicated as green fluorescence granular with diffuse distribution in cytoplasm, while cellular wall images exhibited red fluorescence upon being stained with Alexa-555 WGA. The combination of smFISH method and super-resolution microscopy techniques provides the potential clues and technical reference for further studies on revealing sRNA localization and sRNA-mediated regulatory mechanism.