Aims: The aim of this study was to identify the gene responsible for degradation of toxic allelochemicals of Parthenium by generating Tn5-induced mutant of Pseudomonas putida NBRIC19. Furthermore, the study characterizes the mutant at physiological, biochemical and molecular level that helped in understanding the mechanisms of reducing the allelopathic inhibition of Parthenium by Ps. putida NBRIC19. Methods and Results: Tn5 mutant S-74.3 showing inability to degrade toxic allelochemicals was selected after screening 22 000 transconjugants. Tn5 flanking SucB gene (dihydrolipoamide succinyltransferase) of Ps. putida NBRIC19 was found to be responsible for the degradation of toxic allelochemicals that also affected biofilm formation, chemotaxis and alginate production under toxic environment of allelochemicals. Phenotypic microarray data revealed that the respiratory activity of Ps. putida NBRIC19 and S-74.3 differed on 47 substrates including amino acids, carboxylic acids, peptides and some chemical inhibitors. Conclusions: Study revealed that SucB gene regulates processes either directly or indirectly in Ps. putida NBRIC19, which on inactivation made the mutant less compatible for tolerating stress. Significance and Impact of the Study: This work provides the first evidence for a functional role of Ps. putida SucB gene in degradation of toxic allelochemicals of Parthenium that lead to reversal of plant growth inhibition by these toxic allelochemicals. The investigation also revealed interesting features about the involvement of microbes in plant-plant allelopathic interactions.