Water deprivation can be a major stressor to microbial life in surface and subsurface soil. In unsaturated soils, the matric potential (Ψm) is often the main component of the water potential, which measures the thermodynamic availability of water. A low matric potential usually translates into water forming thin liquid films in the soil pores. Little is known of how bacteria respond to such conditions, where, in addition to facing water deprivation that might impair their metabolism, they have to adapt their dispersal strategy as swimming motility may be compromised. Using the pressurized porous surface model (PPSM), which allows creation of thin liquid films by controlling Ψm, we examined the transcriptome dynamics of Pseudomonas putida KT2440. We identified the differentially expressed genes in cells exposed to a mild matric stress (–0.4 MPa) for 4, 24, or 72 h. The major response was detected at 4 h before gradually disappearing. Upregulation of alginate genes was notable in this early response. Flagellar genes were not downregulated, and the microarray data even suggested increasing expression as the stress prolonged. Moreover, we tested the effect of polyethylene glycol 8000 (PEG 8000), a nonpermeating solute often used to simulate Ψm, on the gene expression profile and detected a different profile than that observed by directly imposing Ψm. This study is the first transcriptome profiling of KT2440 under directly controlled Ψm and also the first to show the difference in gene expression profiles between a PEG 8000-simulated and a directly controlled Ψm.