The mycobacterial cell wall consists of a core composed of peptidoglycan linked to the heteropolysaccharide arabinogalactan, which in turn is attached to mycolic acids. A variety of free lipids complements the mycolyl residues, whereas phosphatidylinositol mannosides (PIMs), lipoarabinomannan and proteins are interspersed in this framework. As a consequence, the cell envelope is extremely rich in lipids and early work has shown that the lipid content may vary with environmental conditions. To extend these studies, the influence of growth temperature on cell envelope components in Mycobacterium thermoresistibile, a temperature-resistant mycobacterial species, was investigated. Mycolic acid synthesis was reduced at 55 degrees C compared to 37 degrees C and the production of fatty acids, presumably precursors of mycolic acids, was increased. Since fatty acids are elongated by the type II fatty acid synthase complex and consequently by a mycobacterial beta-ketoacyl acyl carrier protein synthase (KasA), leading to mycolic acids, the expression level of KasA was analysed by Western blotting. KasA expression was significantly decreased at 55 degrees C over 37 degrees C. Important changes in the mycolic acid composition were observed and characterized by reduced levels of cyclopropanation and the concomitant accumulation of the cis-olefin derivatives. In addition, striking differences involved in complex lipid composition, including acylated trehaloses and trehalose dimycolate (TDM) were also observed. At 55 degrees C, M. thermoresistibile produced less TDM than at 37 degrees C, which could be explained by the down-regulation of antigen 85 (Ag85) expression as shown by Western blotting. The Ag85 complex represents a family of proteins known to catalyse the transfer of mycolates to trehalose, thereby generating TDM. Furthermore, at 55 degrees C the level of phosphatidyl-inositol hexamannoside (PIM(6)) synthesis, but not that of other PIM species, was dramatically reduced. This observation could be correlated to a decrease of mannosyltransferase activity associated with membranes prepared from cells grown at 55 degrees C as compared to 37 degrees C. Altogether, this study suggests that mycobacteria are capable of inducing important cell-wall changes in response to temperature variations, which may represent a strategy developed by the bacteria to adapt to environmental changes.