A previous study showed that overexpressing protein UnkG decreased the ability of the plant growth-promoting bacterium Pseudomonas putida UW4 to facilitate plant growth and an unkG knockout mutant of P. putida UW4 displayed increased plant growth promotion. When activities of wild-type and the UnkG overexpressing strain, including growth rates, carbon utilization, cell size, 3-indoleacetic acid production, and 1-aminocyclopropane-1-carboxylate deaminase activity, were measured, there were no apparent differences between the strains. Monitoring proteome-level changes to the wild-type and overexpressing transformant by means of two-dimensional difference in-gel electrophoresis followed by mass spectrometry identification of the altered proteins, 1839 protein spots were detected and 16 of the 84 protein spots with changed expression levels were identified. Proteins with increased expression included arginine deiminase, dihydrodipicolinate synthase, azurin, flavoprotein (alpha-subunit), ferredoxin-NADP reductase, ATP-dependent Hs1 protease (ATP-binding subunit), UDP-N-acetyl muramate-l-alanine ligase, biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase, and Fis two-component transcriptional regulator. Proteins with decreased expression included glutaminase-asparaginase, arginine/ornithine ABC transporter, cell division protein FtsZ and glutamyl-tRNA synthetase. The functions of three of the 16 proteins could not be identified. The results are consistent with UnkG being detrimental to plant growth because it acts as a regulatory protein that negatively affects several key cellular functions related to the energy balance of the bacterium.