Doctor of Philosophy
Microbiology & Immunology
S. G. Bradley
Deoxyribonucleic acid (DNA) analyses were used to assess on a molecular level, the relationships among representatives of the genera Streptomyces, Nocardia and Mycobacterium. The methods developed in this study have been used for routine analysis of DNA from a large number of actinomycetes and have given reliable and reproducible data.
DNA isolated from various actinomycetes was characterized by buoyant density determinations in CsCl from which the mole fraction guanine plus cytosine (GC) content was calculated. All the streptomycete DNA preparations studied had buoyant densities in the range of 1.7287 to 1.7312 g cm-3 which corresponded to GC compositions of 70% to 73% GC respectively. The nocardial DNA preparations tested fell in two groups, one with a GC content in the range of 62 to 64% GC and another in a 68 to 70% GC group. The mycobacterial DNA tested had GC values overlapping those of the nocardial DNA specimens; moreover, mycobacterial DNA exhibited a bimodal clustering of GC values, 64 to 65% GC and 67 to 70% GC. All DNA preparations examined by equilibrium buoyant density centrifugation in CsCl contained a single component with no satellite bands.
The method of Warnaar and Cohen for assay of DNA/DNA reassociation on membrane filters was modified for studying reassociation of DNA in high GC organisms, DNA isolated from selected actinomycetes was tested for homology with Streptomyces venezuelae S13 mycelial DNA by direct reassociation experiments . Unlabeled DNA from the various actinomycetes was immobilized on Schleicher and Scheull nitrocellulose B-6 membrane filters and then incubated for 15 to 20 hr at 70 C with 14c-labeled DNA. The measure of relatedness was the relative percentage of renaturation of a denatured test DHA with labeled, dentured homolocous DNA. Unrelated DNA having GC contents of 50 and 70% were included as controls. The streptomycetes studied were relatively homogeneous in that measurable interspecific duplexes were formed between the reference DNA and all streptomycete DNA examined. Significantly, the results also suggested that S. venezuelae S13 was related to the nocardial specimens examined but was not related to the mycobacterial cultures studied. The results agreed generally with prior agar-gel studies on DNA reassociation and with previous classifications.
Nucleotide sequence divergence in DNA extracted from streptomycetes and nocardiae was determined by measuring the extent of renaturation at 60 C and 70 C. The use of thermal elution of labeled, renatured duplexes from filters substantiated the existence of a class of nucleotide sesquences which can reassociate at 60 C but cannot reassociate at the more exacting 70 C incubation temperature. The use of exacting incubation conditions (70 C) permitted the formation only of t hose DNA duplexes that exhibited a high degree of thermal stability and hence, closely related to the reference DNA. The non-exacting 60 C incubation allowed those sequences to associate which were distantly related. The ratio of binding at 70 C to the binding at 60 C was designated the Divergence Index (DI). The DI was useful for gauging the presence or absence of closely related genetic material and for determining divergence patterns. The conclusions obtained from this method were corroborated by the much more time consuming thermal elution method. The divergence studies suggested that the streptomycetes contain a wide spectrum of related sequences compared to the reference DNA. Interestingly, the nocardiae examined seemed to have a small but significant amount of conserved nucleotide sequence compared to the S. venezuelae Sl3 reference.
During these studies on actinomycete DNA it was realized that DNA from S. venezuelae S13 spores had novel properties. As spores aged the buoyant density in CsCl decreased from 1.727 to 1.707 g cm-3, the midpoint of thermal denaturation (Tm) in 0.1 x SSC increased from 85 to 88.5 C, and the apparent reassociation with mycelial DNA decreased from 100 to 30% . Spore DNA in 5 M NaCl04 had the same Tm as mycelial DNA. Spore DNA (1.707 g cm-3 ) after heat denaturation showed a single band in CsCl (1.722 g cm-3). Spore DNA was resistant to pancreatic deoxyribonuclease I, but became progressively sensitive after treatment with 0.5 M sodium acetate. Chemical nucleotide analysis of spore and mycelial DNA showed no detectable difference in GC content. The aberrant nature of spore DNA was not affected by pronase or ribonuclease. Washing the spores with ethanol and acetone prior to DNA extraction restored the isolated DNA to normal buoyant density and Tm values. When alcohol and chloroform extracts of spores were dried and mixed with authentic DNA preparations, no change in Tm or buoyant density was found. Spore DNA was yellow in color at pH values below 12 and pink in color at values above 12. Spore DNA heated in high salt showed three characteristic peaks in Sephadex G-100 column chromatography, but only one peak was found with comparably treated mycelial DNA . Attempts to characterize these peaks have been inconclusive to date. Chemical analyses of spore DNA showed 15 to 20% Folin positive material, 40 to 50% more phosphorus than mycelial DNA and no detectable sugars. It appeared that whatever was bound to the spore DNA could be partitioned to added DNA. Experiments of this type were successful only if the test DNA was added to freshly disrupted, dehydrated spores with crushed dry ice. Rehydration of spores showed loss of this binding activity. No chromatographically identifiable compounds or characteristic colors were extracted from spore DNA by a number of solvents and conditions including: acetone, ethanol, ethanol-ether, ethyl acetate, butanol, 8 M urea, 5% cold trichloroacetic acid and 1 N KOH at 100 C for 30 min.
A crude pigmented fraction was isolated from spores which had similar chemical characteristics as aberrant spore DNA. This pigment could not be demonstrated in mycelia; moreover, pigment production seemed to be directly correlated with the age of the spores. The data suggested that this pigment is probably bound to spore DNA and is responsible for the aberrant characteristics of S. venezuelae S13 spore DNA.
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