Mycobacterium sp. strain KMS was isolated from soils where remediation of polycyclic aromatic hydrocarbons was active. This isolate is a competent plant root colonizer through utilization of an array of carbon substrates available in the root exudates. Bioinformatic analyses based on the KMS genome propose pathways for C4- and C3-intermediate conversions during growth of the isolate on substrates requiring gluconeogenesis. Expression of candidate genes for these pathways was compared using semi-quantitative RT-PCR from cells grown on acetate, succinate, benzoate, or pyrene as sole carbon sources requiring gluconeogenesis during growth. Expression was examined for cells grown on fructose and mannitol, where gluconeogenesis would not be essential. Transcript accumulation in cells grown on all the carbon sources confirmed expression from genes involved in the glyoxylate shunt and a gene encoding a novel enzyme to complete the tricarboxylic acid cycle, a membrane-associated malate:quinone oxidoreductase (MQO). Transcript accumulations for genes encoding phosphoenolpyruvate carboxykinase, malic enzyme, and phosphoenolpyruvate synthase were weak for mannitol growth but were detected for the other carbon sources. Activities for PEP synthase and the membrane-associated MQO were confirmed in cell extracts at different levels indicating feasibility of their function in production of PEP for gluconeogenesis in this soil Mycobacterium.