Ultrastructure of Nocardia-like variants of Mycobacterium smegmatis and chemical composition of the basal cell wall layer

Abstract

Mycobacterium smegmatis, its orange-red--pigmented (OR) variants, and back mutant strains were examined by electron microscopy using ultrathin sectioning, negative or positive staining, and freeze-fracture-etching methods. The parental and back mutant strains showed almost identical ultrastructures. Specifically, thick ramified fibers measuring about 15 nm in diameter were always visible in the positively stained cell wall, although they were not readily visualized with negative staining or freeze-fracture-etching. In contrast, the cell walls of OR variants contained fibrous networks measuring about 11 nm in diameter, which could be observed by positive and negative staining as well as freeze-fracture-etching. Although cytoplasmic structures appeared similar among the four strains examined, mesosomes were significantly more abundant in the OR variants. The basal layer of the cell wall obtained as a phenol residue consisted of a dense membranous matrix containing scattered fibrous structures in the parental and back mutant strains, and fibrous networks in the OR variants. Chemical analyses showed that the basal layers of all four strains contained the same neutral sugars, amino sugars, and amino acids, i.e., arabinose, galactose, muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid. The alpha-branched, beta-hydroxylated fatty acids contained in the basal layers differ among the four strains, however, with nocardomycolic acids being present in the OR variants and mycolic acids in the parental and back mutant strains. Our previous conclusion that OR variants of M. smegmatis have characteristics similar to those of nocardia is supported by the present study.