Gross transcriptomic analysis of Pseudomonas putida for diagnosing environmental shifts

The biological regime of Pseudomonas putida (and any other bacterium) under given environmental conditions results from the hierarchical expression of sets of genes that become turned on and off in response to one or more physicochemical signals. In some cases, such signals are clearly defined, but in many others, cells are exposed to a whole variety of ill-defined inputs that occur simultaneously. Transcriptomic analyses of bacteria passed from a reference condition to a complex niche can thus expose both the type of signals that they experience during the transition and the functions involved in adaptation to the new scenario. In this article, we describe a complete protocol for generation of transcriptomes aimed at monitoring the physiological shift of P. putida between two divergent settings using as a simple case study the change between homogeneous, planktonic lifestyle in a liquid medium and growth on the surface of an agar plate. To this end, RNA was collected from P. putidaKT2440 cells at various times after growth in either condition, and the genome-wide transcriptional outputs were analysed. While the role of individual genes needs to be verified on a case-by-case basis, a gross inspection of the resulting profiles suggested cells that are cultured on solid media consistently had a higher translational and metabolic activity, stopped production of flagella and were conspicuously exposed to a strong oxidative stress. The herein described methodology is generally applicable to other circumstances for diagnosing lifestyle determinants of interest. 100 microL of an over night culture of Pseudomonas putida KT2440 was cultured in M9 minimal media supplemented with 0.2 % (v/w) citrate in the following conditions: 5 mL of liquid media during 6 hours, 12 hours or 24 hours incubation, and solid media (25 mL of 1.5% (v/w) agar plate) during 6 hours, 12 hours or 24 hours incubation. RNA was collected and sent for RNASeq service in two biological replicates per condition. Each time point ( 6, 12 and 24 hours) were used to contrast results for liquid and solid media in order to check what genes are transcribed differentially in cells exposed to a solid surface vs cells that live in a liquid media.