Analysis of dissolved oxygen levels The measurement of the dissolved oxygen (DO) concentration of 50 and 150 rpm cultures was performed using a Knick KNI913 oxygen meter. DO levels were measured during culture, at 15 min intervals for 24 hours. Environmental stress assays The assessment of cell Anlotinib viability following exposure to saline, acid and thermal stress was performed on P. putida KT2440 grown at 50 and 150 rpm for 15 hours as described previously [38]. The concentrations of each stress agent were as follows: 5% NaCl for osmotic stress and 10-4 M citric acid for acid stress resistance (pH = 5). For heat shock, exposure of cultures to a temperature of 55°C was applied.

Cells were exposed to each stress for 30 minutes. Bacteria were diluted and MLN2238 molecular weight plated on LB agar before and after exposure to the stress factors in order to determine the survival percentage. Bacterial morphology The morphology of P. putida KT2440 following incubation at different shaking speeds was visualized

by fluorescence microscopy of Hoechst stained cells. Briefly, 600 μl of bacterial culture (after 15 hours of growth) was resuspended in 500 μl 70% ethanol to fix GS-4997 cell line the cells, incubated at room temperature for 20 min and resuspended in saline solution. Next, 2.5 μl Hoechst solution (200 μg/ml) (Hoechst 33258, Sigma-Aldrich, Belgium) was added and incubated for 20 min. Five microliters of this suspension was transferred to a microscopic glass slide, covered with a coverslip and analyzed with a Zeiss Axiovert 100M fluorescence microscope (350 nm filter, 100x oil objective). Acquisition of images was performed with an Axiocam and further processed using the Axiovision software package. Flow cytometry analysis P. putida KT2440 grown at different shaking speeds was analyzed with an Accuri C6 flow cytometer (Accuri Cytometers) to assess the average cell length. Forward and side scatter signals were measured and a total eltoprazine of at least 10,000 cells were recorded for each sample. The respective cell populations were delimited to eliminate background signals originating from cell debris. All data analysis was performed with the CFlow Software. Proteomics Protein

extraction and analysis was performed on P. putida grown at 50 and 150 rpm for 15 hours. Proteins were extracted and labeled isotopically using ICPL, and the post-digest procedure was performed as described in [39]. Labeled tryptic peptides were submitted to online 2D-LC separation prior to MS/MS analysis as described previously [39], except that SCX column was eluted with 11 plugs of increasing NH4Cl concentration (5, 10, 25, 50, 75, 100, 125, 150, 200, 400 and 800 mM in loading solvent). For MS/MS data processing, peptide peaks were detected and processed using Mascot Distiller (version 2.3.2). Created peak list was used as the input for Mascot MS/MS Ions searches using an in-house Mascot 2.2 server (Matrix Science) against the NCBInr database restricted to Pseudomonas putida (KT2440).