RNA-Seq and data analysis

For RNA-Seq comparing PAO1 WT, PAO1 alpA(stop), and PAO1 ∆alpA mutant cells, overnight cultures of biological triplicate samples were back-diluted to OD₆₀₀ of 0.01 in 3 mL of LB and grown to mid-log-phase (OD₆₀₀ of ~0.5). Ciprofloxacin was then added to cultures at a final concentration of 1 µg/mL and 1 mL of each culture was harvested 120 min later. TriReagent was used for RNA isolation (Molecular Research Center). RNA isolation was performed using Zymo Direct-zol RNA Miniprep Plus kit according to kit instructions. RNA was then sent to the Broad Institute for library prep and sequencing or was made into cDNA for qRT-PCR analysis. RNA-Seq libraries were constructed and sequenced at the Broad Institute of MIT and Harvard by the Microbial ‘Omics Core and Genomics Platform, respectively. Sequencing reads from each sample were demultiplexed based on their associated barcode sequence using custom scripts. Reads were aligned to the PAO1 genome ({"type":"entrez-nucleotide","attrs":{"text":"NC_002516","term_id":"110645304","term_text":"NC_002516"}}NC_002516) using BWA (version 0.7.15)⁵³ and read counts were assigned to genes and other genomic features using custom scripts. Differential expression analysis was conducted with DESeq2 (ref. ⁵⁴).

For RNA-Seq experiments comparing PAO1 containing plasmid pAlpA to PAO1 containing plasmid pPSV38 (the empty vector control; referred to as pEV) and PAO1 ∆relA containing plasmid pAlpA to PAO1 ∆relA containing plasmid pPSV38, biological triplicate samples of cells were back-diluted from overnight cultures to an OD₆₀₀ of 0.01 in 200 mL of LB supplemented with 30 µg/mL gentamicin and grown at 37 °C with shaking for 2 h (to an OD₆₀₀ of ~0.04). IPTG was then added at a final concentration of 1 mM and cells were grown for another 90 min to mid-log-phase (i.e. an OD₆₀₀ of ∼0.3–0.4). Specifically, the OD₆₀₀ of the cultures used for RNA isolation were as follows: WT with pAlpA (0.36–0.38), WT with pEV (0.36–0.42), ΔrelA with pAlpA (0.3–0.34), and ΔrelA with pEV (0.3–0.4); 2 mL of cells were harvested by centrifugation at 3200×g for 10 min. RNA was isolated by resuspending cells in 1 mL of TriReagent (Molecular Research Center) and cells were lysed by incubation at 60 °C for 10 min⁵⁰. Supernatants were extracted with 200 µL of chloroform, RNA was then precipitated with ethanol, pelleted by centrifugation at 21,000×g and washed with 75% ethanol⁵⁰. Following resuspension in water, RNA was treated with RNase-free DNase I (Promega) for 1 h at 37 °C, then purified through a second round of treatment with TriReagent and chloroform⁵⁰. Following a final ethanol precipitation step, RNA was resuspended in water. RNA-Seq libraries were made by first depleting ribosomal RNA from 5 µg of total RNA using the Ribo-Zero Magnetic Kit (Bacteria) (Epicentre) according to the manufacturer’s specifications. The remaining RNA was ethanol-precipitated, then used to generate RNA-Seq libraries using the NEB-Next Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s protocol. Libraries were size-selected by PAGE using a 5% gel with TBE (Biorad), stained with SYBR gold nucleic acid stain (Life Technologies), and visualized using a blue-light transillumination system. Fragments corresponding to 150–300 bp were gel purified, ethanol-precipitated, and resuspended in TE buffer according to the NEBNext Multi-plex Small RNA Library Prep Kit for Illumina (New England Biolabs) protocol⁵⁰. RNA quality was determined by Agilent Bioanalyzer. Libraries were sequenced by Elim Bio-pharmaceuticals, Inc. (Hayward, CA), using an Illumina HiSeq 2500 which generated 50 bp single-end reads. Reads were aligned to PAO1 genome ({"type":"entrez-nucleotide","attrs":{"text":"NC_002516","term_id":"110645304","term_text":"NC_002516"}}NC_002516) using Bowtie2 (ref. ⁵⁵). Differential expression analysis was conducted with DESeq2 (ref. ⁵⁴).