Rna bacteria protect reagent

Total RNA was isolated from the P. aeruginosa wild type or mutant strains as formerly explained (Ducret et al., 2020 (link)). Briefly, overnight cultures were diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 and grown for 2 h 30 min in M-LB containing 30 μM TPEN. 0.5 mL of each culture was mixed with 1 mL of RNA Bacteria Protect Reagent (Qiagen) immediately after 2 mM ZnCl₂ induction (t0) and after several time points as indicated in the different figures. Total RNAs were extracted with an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. 5 μg of total RNA were treated with RQ1 RNase-free DNAse (Promega) for 2 h at 37°C, followed by phenol/chloroform extraction and ethanol precipitation.

The prototypic type A virulent strain, F. tularensis subsp. tularensis SCHU S4 was grown routinely on blood cysteine glucose agar (BCGA) containing 4 % cysteine, 4 % histidine, 5 % glucose and 10 % fresh-filtered defibrinated horse blood. Bacterial growth from a freshly streaked plate was used to inoculate 50 ml cultures CDM without dl-serine. Where required, CDM was supplemented with 1 or 10 µg ml⁻¹ serine hydroxamate to induce the stringent response. Cultures were grown aerobically at 37 °C with shaking at 250 r.p.m. for 6 h. At this point OD₆₀₀ was measured and 1 ml of bacterial culture removed, serially diluted and 100 µl spread onto BCGA plates to enumerate bacteria. Following overnight incubation 2 ml bacterial culture were frozen down in RNA Bacteria Protect reagent (Qiagen) at a ratio of 2 : 1 (RNA Protect reagent: culture) for subsequent RNA extraction. Total RNA was extracted according to the Qiagen RNeasy kit instructions, and an on-column DNase digestion was performed. All total RNA extracts were diluted 1/100 and assessed for concentration and quality on the Agilent Bioanalyzer, according to the manufacturer’s instructions for the RNA mRNA pico chip protocol (Agilent Technologies). All work undertaken with Francisella strains was performed in a containment level III laboratory in accordance with relevant legislative requirements.

Cell samples from bacterial co-cultures with tomato roots were extracted as follows: the suspension was filtered with a 20 μm Steriflip filter (Millipore Corp.) to eliminate all plant debris. Filtrate was centrifuged at 8000 × g and pellet was collected and suspended in RNA Bacteria Protect Reagent (Qiagen Cat. 76506) following the manufacturer’s instructions. Prepared samples were stored at −70°C for further processing. Total RNA was extracted from the stored samples as per Jahn et al. [57 (link)]. Quantity of extracted RNA was measured using the Nanodrop 2000C spectrophotometer. Quality was assessed on denaturing electrophoresis gels. Purified RNA was reverse-transcribed to cDNA using the SuperScript® Vilo^TM cDNA Synthesis Kit (LifeTechnologies Cat. 11754–050), according to manufacturer’s instructions. Quality and quantity of cDNA were assessed by spectrophotometry (Nanodrop Technologies, Inc.).