Genetic and chemical control of tuberculostearic acid production in subspecies .

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. subspecies () growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including . While C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for .IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for . This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of subspecies produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of . This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.