Using RNA Seq to investigate adaptation mechanisms in P. aeruginosa

We demonstrate that the versatile environmental bacterium Pseudomonas aeruginosa adapts a virulence phenotype after serial passage in Galleria mellonella as an invertebrate model host. The virulence phenotype was not linked to the acquisition of genetic variations and was sustained for several generations, despite cultivation of the ex vivo virulence-adapted P. aeruginosa cells under non-inducing rich medium conditions. Transcriptional reprogramming seemed to be induced by a host-specific food source as reprogramming was also observed upon cultivation of P. aeruginosa in medium supplemented with polyunsaturated long-chain fatty acids. Methods : mRNA profiles were generated for Pseudomonas aerugionsa samples derived from LB-cultures grown to an OD600 =2. The removal of ribosomal RNA was performed using the Ribo-Zero Bacteria Kit (Illumina) and cDNA libraries were generated with the ScriptSeq v2 Kit (Illumina) . The samples were sequenced in single end mode on an Illumina HiSeq 2500 device and mRNA reads were trimmed and mapped to the NC_008463.1 (PA14) reference genome from NCBI using Stampy pipeline with defaut settings. Overall design: Isolate CH2658 was subjected to in vivo and in vitro evolution experiments in this study. This isolate was obtained from the lab of G. Gastmeier, Charite Berlin, Germany. The in vivo passages (using G. mellonella) are named CH2658 I-IV corresponding to passages 1 4. The last passage CH2658 IV corresponds to the “evolved strain” and was passaged in LB (four days, two passages a day) to generate revertants which are referred to as CH2658 Rev1-4 corresponding to samples from day1-4. The last passage CH2658 Rev4 is called “revertant”. Additionally, the clinical isolate was passaged under in vitro conditions in the presence of linolenic acid (Roth) with (CH2658 Lil+P) and without paraffin (CH2658 Lil). As controls, CH2658 was passaged in LB (CH2658 LB) and in LB supplemented with paraffin (CH2658 LB+P). The in vitro passage experiment was conducted for four days and two passages a day.

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Kordes A, Grahl N, Koska M, Preusse M, Arce-Rodriguez A, Abraham WR, Kaever V, Häussler S