Evolution of biofilm-adapted gene expression profiles in lasR-deficient clinical Pseudomonas aeruginosa isolates

Purpose : The goal of this study was to use RNA Seq to explore the success of lasR mutants in the opportunistic pathogen Pseudomonas aeruginosa in a clinical context by profiling the functional consequences of patho-adaptive mutations in clinical isolates. Methods : mRNA profiles were generated for Pseudomonas aerugionsa samples by deep sequencing.Ribosomal RNA was removed by using the RiboZero Bacteria kit (Illumina). cDNA libraries were synthesized using the SMARTScribe Reverse Transcriptase (Takara) followed by a PCR enrichment using the AccuPrime HiFi Taq polymerase (Invitrogen). Enzymatic reactions were carried out in the presence of SUPERase·In™ RNase Inhibitor (Invitrogen); RNACleanXP beads (Agencourt) were used for all RNA purification steps. Quality checks were performed before, during and after cDNA library preparation with the RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq 6000 (paired-end mode; 2 x 50 bp) and mRNA reads were trimmed using the tool ‘cutadapt’ (version 3.5) with customized settings and mapped to the NC_008463.1 (PA14) reference genome from NCBI using ‘bowtie2’ (version 2.3.5.1) with the settings “--very-sensitive-local; --no-mixed; --fr; --no-unal”. mRNA profiles from Pseudomonas aeruginosa derived from cells that were either grown in LB to an OD600 =2 (planktonic) or inoculated at a starting OD600 of 0.002 in LB and grown statically in half-area 96-well µClear microtiter plates at 37 °C in humid atmosphere for 48 h and deep sequenced using Illumina NovaSeq6000.