Base Composition of Deoxyribonucleic Acid Isolated from Mycobacteria

Lawrence G. Wayne, Wendy M. Gross
1968 Journal of Bacteriology  
Guanine plus cytosine values of deoxyribonucleic acid derived from 30 cultures representing 14 mycobacterial species or varieties are presented. These data provide impressive reasons for maintaining the separation between the genera Corynebacterium and Mycobacterium; no conclusions can be arrived at from these data with respect to the Nocardia-Mycobacterium relationship. A bimodal clustering, in terms of guanine plus cytosine composition, is apparent within the genus Mycobacterium. In general,
more » ... erium. In general, all members of any single phenetic species appear to fit into one or another of these clusters. The phenetic separation of species is, in some cases, confirmed by separation in terms of guanine plus cytosine values. The bimodal separation of guanine plus cytosine values within the genus Mycobacterium does not correspond to a division of the species into slow and rapid growers; it thus provides no justification for splitting Mycobacterium into two genera, composed of slow and rapid growers. This is not to say that such a split would not be useful, only that these data do not contribute to such a decision. Any further attempts to correlate phenetic classification with properties of mycobacterial deoxyribonucleic acid will require more specific techniques, such as molecular hybridization. The principles of numerical taxonomy have been applied to the study of the members of the genus Mycobacterium by a number of investigators in recent years. It is generally recognized that conventional bacteriological tests provide little help in resolution of species within the genus Mycobacterium, and it has become necessary to develop specialized schemes of biochemical testing in order to provide data which may be used for taxonomic studies within this genus (11). One of the consequences of the use of specialized schemes within a genus is the difficulty encountered in attempting to relate the different levels of taxonomic subdivision within any one genus to those recognized in another genus. It is therefore desirable to attempt to correlate results of numerical taxonomic studies with determination of base composition and, eventually, of molecular homology of the deoxyribonucleic acid (DNA) derived from different members of the genus under investigation. This is a report of the base composition of DNA isolated from representatives of a number of mycobacterial species. MATERIALS AND METHODS Cultures from the stock culture collection in this laboratory are maintained on Lowenstein-Jensen egg medium and transferred twice a year. The bacteria were grown in a shallow layer of liquid Sauton medium over Sauton agar (13). The thick slurry of bacterial cells so produced was aerated vigorously on a magnetic stirrer for 3 days and abruptly subjected to anaerobic conditions in the presence of sodium ethylenediaminetetraacetate (EDTA) and the proteolytic enzyme Pronase; 24 hr later, the slurry was exposed to 0.5% sodium desoxycholate at 56 C for 90 min. The extract was then deproteinized with phenol; the DNA was precipitated with ethyl alcohol, redissolved and treated with pancreatic ribonuclease, and finally deproteinized again and reprecipitated with ethyl alcohol. Details of this method of isolation of mycobacterial DNA and some characterization of the properties of the material so produced have been described previously (13). The guanine plus cytosine (GC) values were calculated from the melting temperatures (Tm) by plotting hyperchromic melting curves. DNA was dissolved to a concentration of approximately 20 gg/ml in the 10-2 M sodium phosphate, 10-3 M EDTA buffer at pH 7.0 (Bohacek et al., 1). The solutions were transferred to glass-stoppered cuvettes and placed in a Beckman DU spectrophotometer equipped with thermospacers on each side of the cuvette holder. The thermospacers adjacent to the cell holder were connected to a circulating hot water-ethylene glycol bath, the temperature of which was raised at a rate of 1 C per 10 min beginning at 74 C. Temperature was measured by placing a thermometer directly into one of the cuvettes with a light shield placed over it. The per cent GC was calculated according to the equation (1): Tm = 51 + 0.45 (GC). Results obtained with this method were compara-1915 on May 9, 2020 by guest
doi:10.1128/jb.96.6.1915-1919.1968 fatcat:y7jzribtzjgl7cwyl6pgqg5wdm