Effect of AHL-QS on protein expression in P. aeruginosa PAO1-UW
We found that several Pseudomonas aeruginosa strains accumulate N2O at higher cell densities. This is caused by quorum sensing (QS) and not regulated on the transcriptional level. To investigate whether this is a posttranscriptional or posttranslational effect we collected cells for proteomics measurements from P. aeruginosa PAO1-UW wild type (wt1-4)and a rhlI/lasI knockout mutant (dm 1-4)during the transition to denitrification. The mutant served as a AHL-QS negative control since it lacks the enzymes for the production of these specific signaling molecules. Comparing the expression of genes in the nos operon, which encode the structural and ancilliary proteins for N2O reduction, revealed that the restricted N2O redcution in the wild type is not caused by the downregulation of any of these genes, but that it is a posttranscriptional effect. Samples wt1-4 and dm1-4 were used for this comparison. Samples wt 5 and dm 5 were excluded due to poor quality. The text file was generated by analysis with Maxquant.
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Sampling for proteomics Samples for proteomics were taken after N2O or N2 accumulation was observed in wild type and rhlI/lasI PAO1-UW respectively. Vials sampled for nitrite measurements were not sampled for proteomics.The content of entire vials was transferred to centrifuge tubes and centrifuged at 10000 g for 20 min at 4°C. The pellets of each biological replicate were pooled and stored at -20°C until proteins were extracted from the sample. Protein extraction and clean up The protein pellet was resuspended in 500 µL lysis buffer (4% SDS, 50 mM Tris-Cl, 10 mM DTT) and heated to 95°C for 10 min. The solution was added to a FastPrep tube with 200 mg acid washed glass beads (<106 µm). The bead-beating was run thrice at 6.5 m/s for 60 s. After spinning the tubes down, the supernatant was stored at -20°C. Extracted protein was purified with the Suspension trap (STrap) method. The STrap tips were prepared by stacking 4 Empore C18 and 12 Munktell MK360 quartz filters in a 200 µL pipette tip. To each tip, 170 µL STrapping solution (90% methanol with 10 mM Tris, pH 7.1) was added and centrifuged briefly. Samples were diluted with the same volume of lysis buffer containing 20 mM DTT and 20% SDS was added to reach a final concentration of 4% SDS and 10 mM DTT. 17 µL of this solution was denatured at 95°C for 10 min. Iodoacetamide was added to a final concentration of 50 mM and incubated in the dark for 20 min. After acidification with 2 µL 12.15% phosphoric acid, samples were added to the top layer of the Strap solution inside the tip and centrifuged for 10 min at 4000 g or until all of the solution passed through the filter. The filter was washed by adding another 100 µL Strap solution and repeating the centrifugation. Afterwards, 100 µL 50 mM ammonium bichloride was added and the tips were centrifuged again. 12 µL trypsin solution (33 ng/µL in 50 mM ammonium bichloride) was added spun briefly before incubation for 1 h at 47°C. The tips were again centrifuged briefly until the trypsin solution passed through the filters and 100 µL 0.5% trifluoroacetic acid (TFA) was added to the flowthrough before transferring it to the tip again and centrifuging as described above. The filter was washed by adding 100 µL 0.1% TFA and repeating the centrifugation before eluting protein from the column with 50 µL elution solution (80% acetonitrile, 0.1% TFA), incubating for 2 min at room temperature and centrifuging the tip for 5 min at 2500 g. The samples were dried in a speedvac and stored at -20°C. Lysates were analyzed as described by Conthe et al. (2019). LFQ and statistical analysis was performed with Maxquant (126.96.36.199) and Perseus (188.8.131.52) respectively (Cox and Mann, 2008, Tyanova et al., 2016).