ABSTRACT: Differential gene expression analysis of Leptospirillum ferriphilum DSM 14647 cells treated with quorum sensing singnal compounds. Briefly, Leptospirillum ferriphilum DSM 14647 cells were grown to early stationary phase in Mackintosh basal salt solution (Mac medium) pH 1.8 (Mackintosh 1978) with 71.6 mM Fe2+ (supplied as FeSO4•7H2O). Cultures were incubated under constant shaking at 140 rpm and 37 °C. Cells were harvested by centrifugation (7.000 g, 10 min). Subsequently cells were inoculated at 10^8 cells/mL in 100 mL Erlenmeyer flasks with 50 mL Mac medium (pH 1.8) and amended with combined DSF (cis-11-methyl-2-dodecenoic acid, CAS 677354-23-3; Sigma) and BDSF (cis-2-dodecenoic acid, CAS 55928-65-9; Sigma) at 2 µM per compound or with a mixture of N-acyl-homoserine-lactones, namely N-dodecanoyl-DL-homoserine lactone (C12-AHL, CAS 8627-38-8, Sigma), N-tetradecanoyl-DL-homoserine lactone (C14-AHL, CAS 98206-80-5, Sigma), N-(3-hydroxydodecanoyl)-DL-homoserine (3-OH-C12-AHL, CAS 182359-60-0), N-(3-hydroxytetradecanoyl)-DL-homoserine lactone (3-OH-C14-AHL, CAS 172670-99-4) at 5 µM per compound. Controls were supplemented with DMSO as used as solvent for signal compound stock solutions. After inoculation 32 mM iron(II) ions were added. The assay flasks were incubated for two hours at 37 °C and 140 rpm. Assays were periodically sampled for determination of Fe2+ concentrations using the phenantroline method (Harvey, Smart et al. 1955). Within this time the inhibitory effect of DSF/BDSF on Fe2+ oxidation was confirmed and subsequently the flasks were rapidly cooled on ice and by addition of 1 volume ice-cold Mac medium (pH 1.8). The cultures (n = 4 per condition) were centrifuged at 12,500 × g for 10 min at 4° C. The cell pellet was washed twice by re-suspending in 2 mL of sterile, ice-cold Mac medium (pH 1.8) and then flash frozen in liquid nitrogen and stored at -80 °C. The nucleic acid extraction was conducted as described by (Christel, Fridlund et al. 2016) with modifications. Briefly, the cells were re-suspended in lysis buffer (0.02 % sodium acetate, 2 % sodium dodecyl sulfate, 1 mM EDTA, pH 5.5) and lysed using Tri-reagent (Ambion) and the lysate was treated with bromo-chloro propane. Nucleic acids were precipitated with isopropanol, cleaned with 80% ethanol, dried at room temperature and subsequently treated with DNAse I (Thermo Fisher Scientific®). Ribosomal RNA depletion was conducted using the QIAseq® FastSelect™ −5S/16S/23S kit for bacterial RNA samples without fragmentation. Nucleic acid quantification and quality control were assessed by agarose gel electrophoresis, NanoDrop, Qubit™ RNA HS Assay kit (Invitrogen®) and the Agilent 2100 Bioanalyzer . Libraries (12 in total) were prepared by SciLifeLab, Stockholm, Sweden using the Illumina TruSeq stranded mRNA Kit. Paired-end sequencing (2 × 151 bp) was performed on one Illumina NovaSeq6000 lane using 'NovaSeqXp' workflow in 'S4' mode flowcell. Bioinformatics and statistics The Bcl to FastQ conversion was performed using bcl2fastq_v2.20.0.422 from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+. at SciLifeLab, Stockholm, Sweden. The quality of the raw sequencing reads was assessed with FastQC/MultiQC (Andrews 2010, Ewels, Magnusson et al. 2016). Subsequently adapter sequences were removed using Cutadapt/TrimGalore 0.6.1 (Martin 2011, Krueger 2019). Paired-end transcript reads were mapped against the reference genome with the accession number GCA_900198525 (Christel, Herold et al. 2017) using Bowtie2 (Langmead and Salzberg 2012), sorted by their genomic location using the samtools sort function (Liao, Smyth et al. 2019) and counted using feautureCounts of the Rsubreads package (Liao et al. 2019). Raw counts were then processed for assessment of statistically significant differential gene expression with DESeq2 (Love, Huber et al. 2014) by determination of Log2-fold changes (LFC) and corresponding p-values (padj, adjusted p-values).
