Root exudates are chemical compounds that are released from living plant roots and provide significant energy, carbon, nitrogen and phosphorus sources for microbes inhabiting the rhizosphere. The exudates shape the microflora associated with the plant, as well as influences the plant health and productivity. Therefore, a better understanding of the trophic link that is established between the plant and the associated bacteria is necessary. In this study, a comprehensive survey on the utilization of grapevine and rootstock related organic acids were conducted on a vineyard soil isolate which is Pseudomonas mendocina strain S5.2. Phenotype microarray analysis has demonstrated that this strain can utilize several organic acids including lactic acid, succinic acid, malic acid, citric acid and fumaric acid as sole growth substrates. Complete genome analysis using single molecule real-time technology revealed that the genome consists of a 5,120,146 bp circular chromosome and a 252,328 bp megaplasmid. A series of genetic determinants associated with the carbon utilization signature of the strain were subsequently identified in the chromosome. Of note, the coexistence of genes encoding several iron-sulfur cluster independent isoenzymes in the genome indicated the importance of these enzymes in the events of iron deficiency. Synteny and comparative analysis have also unraveled the unique features of D-lactate dehydrogenase of strain S5.2 in the study. Collective information of this work has provided insights on the metabolic role of this strain in vineyard soil rhizosphere.