Genome-wide analysis of targets for post-transcriptional regulation by Rsm proteins in Pseudomonas putida KT2440

Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences that are bound in vivo by Rsm proteins, in order to identify potential regulatory targets for each of them. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to identify 709 targets, 75 of them common to the three proteins. Relevant targets include genes encoding proteins involved in metabolism, transport and secretion, stress responses, signal transduction and regulation, and the turnover of the second messenger c-di-GMP. The role of Rsm proteins in the utilization of basic amino acids and bacterial fitness in the plant rhizosphere are also exposed. Overall design: Previously constructed derivatives of expression vector pME6032 harboring each rsm gene were used to express His-tagged Rsm proteins in P. putida KT2440. Overnight cultures (10 ml) of KT2440 harbouring each construct were inoculated in 500 ml of LB medium, and incubated at 30°C under shaking to an OD660 of 0.8. At that point, expression of His-tagged-Rsm proteins was induced for 6 h by the addition of IPTG to a final concentration of 0.5 mM. Three biological replicates were run in parallel. Aliquots of 1.5 ml were harvested by centrifugation, instantly frozen with liquid nitrogen and stored at -80?C for total RNA purification. Cells from the remaining cultures were also harvested and cell pellets stored at -80?C. His-tagged Rsm-RNA complex purification were isolated using Ni-NTA Fast Start Kit (Qiagen). Three replicate extractions were done for each culture replica. Total RNA and RNA from Rsm-RNA complex was extracted using RNA isolation kit (Macherey-Nagel) following the manufacturer´s instructions. RNA samples were subsequently treated with RNase-free DNase I (Turbo DNA-free kit, Ambion) to remove DNA traces, as specified by the supplier. Total RNA quality was assessed using Agilent RNA 6000 Nano Kit (Agilent Technologies) in the Agilent 2100 Bioanalyzer. RNA concentration was measured using Qubit RNA BR assay kit (Life Technologies). 1µg of RNA was used for rRNA depletion using Ribo-Zero rRNA Removal Kit (Illumina). One of the biological replicates of RsmA and one of RsmI did not meet the required quality and quantity standards and were discarded.