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Mycobacterium microti walls contained three types of mycolic acids, very similar to those found in Mycobacterium tuberculosis. An alpha-mycolate with two cyclopropane rings, a methoxymycolate with one cyclopropane ring and a methoxyl group, and a ketomycolate with one cyclopropane ring and a keto group were partially characterized. The mycolates made up 34% (by weight) of the peptidoglycan-arabinogalactan-mycolate wall skeleton. Young exponential phase cultures and organisms harvested from mouse lungs contained high proportions of ketomycolates; older cultures had roughly equal proportions of keto- and methoxymycolates. The proportion of alpha-mycolates increased slightly with age of culture, but was always less than one-third of the total.

Isoniazid inhibited C24 and C26 monounsaturated fatty acid synthesis in Mycobacterium tuberculosis H37Ra. Time courses of this inhibition and that ofmycolic acid synthesis were similar.The antimycobacterial drug isoniazid inhibits mycolic acid synthesis in Mycobacterium tuberculosis BCG (10) and H37Ra (5, 9). Mycolic acids, which are a-alkyl f)-hydroxy fatty acids containing up to 90 carbon atoms, are important mycobacterial wall components (1, 2). Isoniazid also inhibits the production of very long-chain (greater than C26) fatty acids other than mycolic acids (8). On the basis of structural analyses (4, 7), it was suggested that these very long-chain fatty acids may be precursors of mycolic acids and that the first reaction specific to mycolic acid synthesis may be the desaturation of tetracosanoic (C24) and perhaps also hexacosanoic acid (C26). We now report that isoniazid inhibits this reaction.M. tuberculosis H37Ra was grown in enriched Middlebrook 7H9 medium (9). Isoniazid was added to 100-ml cultures in early exponential phase (absorbance at 650 nm of 0.1 to 0.2) to give concentrations of 0.5 tLg/ml. Portions (10 ml each) were removed from the cultures immediately before and at various times after the addition of isoniazid. Control cultures had no isoniazid added. Mycolic and nonmycolic fatty acid synthesis was assayed in each portion as described previously (6), except that incubations were for 5 min with 50 ,uCi of sodium [1Y-C]-acetate (57.7 ,uCi/4mol, Amersham/Searle), and bacilli were harvested by filtration through a 0.45-,um filter (Millipore Corp.) and saponified in 2.5 ml of 10% KOH in ethanol-water (1:1, vol/vol) at 900C for 16 h. The fatty acids were fractionated into mycolic acids and short-chain (016 to C19) saturated, long-chain (C24 to CQ ) saturated, C16 to C01 monounsaturated, and C24 to C26 monounsaturated components by thinlayer chromatography, and then the radioactivity in each fraction was determined (3,6,9).Isoniazid, at 0.5 ug/ml, rapidly inhibited the synthesis of C24 to C26 monounsaturated fatty acids, but the synthesis of C24 to C26 saturated fatty acids increased, reaching its maximum at 50 min (Fig. 1).

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