Effect of growth stage on mycolic acid structure in cell walls of Nocardia asteroides GUH-2.

Mycolic acids were extracted from the cell walls of Nocardia asteroides GUH-2 during different phases of growth at 37 degrees C. These were subjected to structural analysis by combining thin-layer chromatography and gas-liquid chromatography with UV and infrared spectrophotometry and mass spectroscopy of both methyl esters and trimethyl silyl derivatives. By analyzing the fragmentation patterns of these derivatives by three different methods of mass spectroscopy combined with gas-liquid chromatographic separation, the different structural subclasses of mycolic acids were quantitated. Significant qualitative and quantitative modifications of specific mycolic acid subclasses occurred in the cell walls of N. asteroides GUH-2 that were growth stage dependent. The mycolic acids that were predominant in the log phase were polyunsaturated (greater than 2 double bonds per molecule), with long chain lengths and even carbon atom numbers (i.e., C54, C56). In contrast, those that were prominent in the stationary phase were more saturated (few or no double bonds) and of shorter overall carbon chain length (less than or equal to C52). Furthermore, stationary-phase cells had significantly increased amounts of mycolic acids with odd-numbered carbon chain lengths (i.e., C49, C51, C53).

Relationship among cell wall composition, stage of growth, and virulence of Nocardia asteroides GUH-2.

The clearance and organ distribution of virulent Nocardia asteroides GUH-2P and the avirulent mutant GUH-2AI at different stages of growth was determined after intravenous inoculation into BALB/c mice. The mutant differed significantly from the parent strain in its ability to survive and grow within the murine host. Since the mutant GUH-2AI had a very different colonial morphology compared with GUH-2P, it was believed that cell surface components might be affected by the mutation that resulted in the loss of virulence. Therefore, cell walls of both GUH-2P and GUH-2AI at different stages of growth were prepared and their composition determined. There were growth-stage-dependent changes in the composition of the cell walls that appeared to correlate with concurrent alterations in virulence; however, the overall chemical composition of the cell wall of the mutant (GUH-2AI) was not significantly different from that of the parent strain (GUH-2P). Both strains demonstrated significant modifications in fatty and mycolic acid composition at different stages of growth. Furthermore, the specific composition of C54 mycolic acids was very different in virulent log-phase cells compared with less-virulent stationary-phase cells, and the avirulent mutant lacked a C54:3 mycolate that was prominent in the virulent log-phase GUH-2P. Thus, C54:3 mycolic acid represented 2.5% of the cell wall (dry weight) in log-phase GUH-2P, but it was undetectable in the walls of GUH-2AI at the stationary phase of growth. These results suggest that certain mycolic acids are associated with virulence.

Purification and properties of a unique superoxide dismutase from Nocardia asteroides.

A unique form of superoxide dismutase was isolated and characterized from Nocardia asteroides GUH-2. This enzyme contains 1 to 2 g atoms each of Fe, Mn, and Zn per mol and exhibits spectral properties suggestive of Fe- or Mn-containing superoxide dismutases. Its Mr = 100,000, and it is composed of four subunits of equal size which are not covalently joined. The amino acid composition of the enzyme was more closely related to the Mn- or Fe-containing enzymes of Mycobacterium species and was least related to the Cu-Zn enzyme of eukaryotes. Azide at 1 and 20 mM inhibits the activity 10 and 41%, respectively, and 5 mM H2O2 inhibits 40%, but 1 or 5 mM cyanide caused trivial effect. The immunofluorescent staining, which was specific for superoxide dismutase of N. asteroides, indicated the association of this enzyme to the outer cell wall of the organism. Further, the enzyme was shown to be selectively secreted into the medium.

Cell wall modification resulting from in vitro induction of L-phase variants of Nocardia asteroides.

The chemical composition of the cell walls of several L-form revertants derived from Nocardia asteroides 10905 was determined at different stages of growth. It was observed that each L-form revertant had a cell well that differed from that of the parental strain when grown under identical conditions. In some strains the peptidolipid and mycolic acid components were affected the most, whereas in other strains the fatty acid, sugar, and mycolic acid moieties were altered. Shifts in mycolic acid size were prominent, whereas the basic peptidoglycan structure appeared to be affected the least. Both the method used to induce the L-form of N. asteroides 10905 and the length of time these organisms were maintained in the wall-less state affected the degree of cell wall modification during the reversion process. Thus, removal of the cell wall appeared to potentiate and select for mutational alterations within the cell envelope of N. asteroides, and these changes resulted in altered cellular and colonial morphology.