Oleaginous microorganisms have considerable potential for biofuel and commodity chem. prodn. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymn. products, accumulated triacylglycerols (TAGs) to 55% of its dry wt. during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis, and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (∼50-fold more abundant than during N-excess). Consistent with Atf8 being a physiol. determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, 3 genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metab. in mycolic acid-contg. bacteria and provide a framework to engineer strains for increased TAG prodn. [on SciFinder(R)]