A novel mycolic acid species defines two novel genera of the Actinobacteria, Hoyosella and Amycolicicoccus Corynebacterineae are characterized by the presence of long-chain lipids, notably mycolic acids (a-alkyl, b-hydroxy fatty acids), the structures of which are genus-specific. Mycolic acids from two environmental strains, Amycolicicoccus subflavus and Hoyosella altamirensis, were isolated and their structures were established using a combination of mass spectrometry analysis, 1 H-NMR spectroscopy and chemical degradations. The C 2 -C 3 cleavage of these C 30 -C 36 acids led to the formation of two fragments: saturated C 9 -C 11 acids, and saturated and unsaturated C 20 -C 25 aldehydes. Surprisingly, the fatty acids at the origin of the two fragments making up these mycolic acids were present in only minute amounts in the fatty acid pool. Moreover, the double bond in the main C 24 aldehyde fragment was located at position v16, whereas that found in the ethylenic fatty acids of the bacteria was at v9. These data question the biosynthesis of these new mycolic acids in terms of the nature of the precursors, chain elongation and desaturation. Nevertheless, they are consistent with the occurrence of the key genes of mycolic acid biosynthesis, including those encoding proteins of the fatty acid synthase II system, identified in the genome of A. subflavus. Altogether, while the presence of mycolic acids and analysis of their 16S rDNA sequences would suggest that these strains belong to the Mycobacteriaceae family, the originality of their structures reinforces the recent description of the novel genera Amycolicicoccus and Hoyosella. INTRODUCTIONThe suborder Corynebacterineae forms a large group of actinomycete species characterized by the presence of specific lipids, notably mycolic acids (a-alkyl, b-hydroxy long-chain fatty acids). The variability of their chain lengths and the complexity of their structures contribute to the definition of the genera, from the simplest corynomycolic acids of Corynebacterium to the most complex and species-specific mycolic acids of Mycobacterium, with intermediate chain-lengths in Rhodococcus, Nocardia and Gordonia (Barry et al., 1998). Recently two coccoidal strains have been isolated from environmental sources, Hoyosella altamirensis, from the Altamira cave in Spain, and Amycolicicoccus subflavus, from saline soil contaminated with crude oil in the Daqing Oilfield of eastern China (Jurado et al., 2009;Wang et al., 2010). By a polyphasic approach, the taxonomic position of the two species has been determined, i.e. Actinomycetales cell wall chemotype IV, menaquinones MK-8, diagnostic phosphatidylethanolamine and tuberculostearic acid. On the basis of 16S rRNA gene sequence analysis, the two species were shown to be most closely related to the genus Mycobacterium (95.6 % similarity with the Mycobacterium fallax type strain for Hoyosella, and 92.7-94.9 % similarity with Mycobacterium spp. for Amycolicicoccus). The two species differ from each other by their DNA G+C content, which is 49...