Time course dependent changes in contents and physical properties of glycolipid species in Rhodococcus rhodochrous

Abstract

The yield of trehalose dimycolate (TDM), the major glycolipid species elaborated by Rhodococcus rhodochrous, a producer of approx. C 40-mycolic acid, was not constant in cells cultured for different periods of time. From cells collected at 24, 36, 72, 144 and 172 h of cultivation the following percentages of TDM in diethyl ether soluble lipids (DESL) were found: 10.8%, 23.4%, 10.0%, 9.0% and 5.0%, respectively. In turn, the cellular content accounted for approx. 0.6%, 1.2%, 0.9%, 0.6% and 0.2%, respectively. On the other hand, the yield of galactose monomycolate (GalMM), a minor glycolipid species maintained at approx. 3.4% in DESL during the different periods of time examined; this value represented about 0.3% of the cellular content. The melting temperatures of TDMs fell between 37°C to approximately 97°C with the lowest value from cells grown for 36 h, whereas the melting temperatures of the GalMMs were in a narrow range between 56°C and 64°C. The methyl ester derivatives of the constituent fatty acid moieties of DTMs and GalMMs migrated on thin layer chromatography like methyl esters of C 40–C 46 mycolic acids, therefore faster than methyl esters of C 28–C 34 mycolic acids but slower than methyl esters of C 50–C 56 mycolic acids. Further analysis of the products of pyrolysis of the methyl ester derivatives of the fatty acid moiety released from TDM after alkaline hydrolysis was carried out using gas chromatography combined mass spectrometry. From these analyses, it was found that the methyl ester derivative of the mycolic acid moiety of trehalose dimycolate from cells grown for 36 h (late exponential phase) displayed the highest degree of unsaturation. This finding was consistent with the low melting temperature of the original glycolipid. In turn, the mycolic acid with the longest chain length of the meroaldehyde subunit (α-subunit) was found in the sample from cells grown for 24 h (early exponential phase).