Significance of Nucleic Acid Hybridization to Systematics of Actinomycetes
S. G. BRADLEY Department of Microbiology, Virginia Commonwealth University, Richmond, Virginia
I. 11. 111. IV. V. VI. VII.
Introduction ...................................... DNA Nucleotide Composition ...................... DNA:DNA Association ............................. The Cot Concept ................................. Optical Reassociation .............................. Phylogenetic Implications ........................... Neutral Mutations ................................. References ........................................
I.
59 60 60
63 64 66 69
70
Introduction
Systematics progresses through three stages: the description of cultures leading to bases for distinguishing between strains; the development of keys, classification schemes, and a system of nomenclature; and finally an assessment of evolutionary relationships. The approaches applied and the techniques used differ for each of these stages. During the descriptive phase, extensive surveys employing diverse tests and observations are needed. During the nomenclatural phase, type cultures must be established as references. During the phylogenetic phase, selected type cultures and representative strains must be examined in detail for evidence about their genetic relationships ( 5 ) . Genetic homologies can be determined by use of the various mechanisms for genetic exchange: transduction, transformation, or syncytic recombination (2). It must be noted, however, that a limited number of genetic determinants can be transferred from one organism to another by episomes or plasmids that are not necessarily homologous with the recipient’s basic genome. In numerical taxonomy, an attempt is made to describe a significant sample of the genome of a group of organisms by analyzing a very large number of overt characteristics. Relationships based upon numerical analysis, therefore, are approximations of the degree of genetic homology in the population examined. Biological tests for homology often fail for reasons other than lack of genetic similarity (2). Accordingly, genetic relatedness can best be measured by comparing the genetic macromolecules themselves. For definitive studies on biological relatedness, genetic homology per se must be determined. Genetic homology is defined in biological terms as the similarity between se59
60
S. G . BRADLEY
quences of loci, but in physical terms, it is the similarity between nucleotide sequences within deoxyribonucleic acid ( DNA) molecules. The genetic potential of an organism is encoded in the linear sequence of the nucleotides in its DNA. These nucleotide sequences determine the sequences of amino acids in structural or enzymic proteins which directly or indirectly constitute the phenotype of the cell. Accordingly, evolutionary divergence from a common ancestor proceeds as the progeny accumulate base substitutions in their DNA. Because of our increased capability to manipulate the DNA molecule, approaches to microbial classification other than traditional determinative systematics can be developed. In fact, an evolutionary approach to bacterial taxonomy, long hindered by lack of an adequate fossil record, now seems feasible. 11.
DNA Nucleotide Composition
The Watson and Crick model of double-stranded DNA predicts that the guanine ( G ) content of a DNA molecuIe equals its cytosine ( C ) content; likewise, the adenine ( A ) and thymine ( T ) contents are also equal. However, the ratio of ( A T ) / ( G C ) , or the mole percent of guaninefcytosine [% G C = ( G + C ) l O O / ( A + T + G + C ) ] may vary from one species to another. As indicated earlier, closely related organisms have very similar nucleotide sequences, and therefore very similar GC ratios. Similarity in % GC does not necessarily indicate genomic similarity but, unlike % GC of DNA from two organisms, establishes that they are not identical. The relationships among selected actinomycetes, with special emphases on streptomycetes, nocardiae, and mycobacteria, have been determined, based upon the nucleotide composition of their DNA (Table I ) . The streptomycetes constituted a homogeneous group whose DNA contained between 69 and 74 mole % guanine cytosine ( % GC). The nocardial and mycobacterial DNA preparations contained 61-69% GC. The nocardial strains fell into either a 62,-64% GC group or a 6749% GC group (22).
+
+
+
Ill. DNA:DNA Association
Because the phenotype of an organism is determined by the nucleotide sequence in its DNA, a comparison of the nucleotide sequences of DNA preparations from two organisms should give a definitive evaluation of their relatedness. Unfortunately, complete direct sequence analysis of a DNA molecule is not currently possible. The complementary nature of the DNA double helix, however, can be used to circumvent these
61 TABLE I NUCLEOTIDE COMPOSITION OF SELECTED ACTINOMYCETE DNA SAMPLES Source
% GCa
Actinomadura Act i n o myces Actinoplanes Ampullariella Dact ylosporangium Micromonospora M ycobacterium M ycococcus Nocardia Oerskovia Planobispora Planomonospora Spirillospora Streptom yces Streptosporangium Streptoverticillium Thermomonospora
68 58 71-73 72 71-73 71-73 64-69 68 61-69 74-76 70-71 72 71-72 69-74 69-7 1 69-7 1 67-69
Values compiled from our published works (4,6,9,14). Q
technical difficulties. It has been established that the two strands of the DNA helix can be separated and specifically reassociated or annealed (19).
The phenomenon of association of complementary strands of DNA provides a powerful tool for exploring the relationships among microorganisms ( 18). Investigators analyzing DNA preparations with 60-70% GC encountered many problems with the earlier hybridization methods (27). Accordingly, my colleagues and I ( 9 , 1 3 ) have modified the method of Warnaar and Cohen ( 2 4 ) for quantitative assay of hybridization between denatured DNA fixed to nitrocellulose membrane filters and free, denatured DNA. We have used this modified technique to assess, on a molecular level, the relationships among representatives of many genera of actinomycetes, in particular Mycobacterium, Nocardia, and Streptomyces (Table 11). The relationships among these organisms are of particular interest because of their special relevance for industry, medicine, and agriculture. Moreover, the taxonomy of the actinomycetes remains a subject for active study and debate. A difficult problem in using nucleic acid association for taxonomic studies is the selection of a system that will give the needed information. There are two general methods to choose between ( a ) association involv-
62
S. G. BRADLEY
TABLE I1 ASSOCIATION OF DNA FIioM Nocardia asteroides STRAINS 300 A N D 330 WITH DNA FROM SICLIK!TEDNOC.\I
S. G. BRADLEY Department of Microbiology, Virginia Commonwealth University, Richmond, Virginia
I. 11. 111. IV. V. VI. VII.
Introduction ...................................... DNA Nucleotide Composition ...................... DNA:DNA Association ............................. The Cot Concept ................................. Optical Reassociation .............................. Phylogenetic Implications ........................... Neutral Mutations ................................. References ........................................
I.
59 60 60
63 64 66 69
70
Introduction
Systematics progresses through three stages: the description of cultures leading to bases for distinguishing between strains; the development of keys, classification schemes, and a system of nomenclature; and finally an assessment of evolutionary relationships. The approaches applied and the techniques used differ for each of these stages. During the descriptive phase, extensive surveys employing diverse tests and observations are needed. During the nomenclatural phase, type cultures must be established as references. During the phylogenetic phase, selected type cultures and representative strains must be examined in detail for evidence about their genetic relationships ( 5 ) . Genetic homologies can be determined by use of the various mechanisms for genetic exchange: transduction, transformation, or syncytic recombination (2). It must be noted, however, that a limited number of genetic determinants can be transferred from one organism to another by episomes or plasmids that are not necessarily homologous with the recipient’s basic genome. In numerical taxonomy, an attempt is made to describe a significant sample of the genome of a group of organisms by analyzing a very large number of overt characteristics. Relationships based upon numerical analysis, therefore, are approximations of the degree of genetic homology in the population examined. Biological tests for homology often fail for reasons other than lack of genetic similarity (2). Accordingly, genetic relatedness can best be measured by comparing the genetic macromolecules themselves. For definitive studies on biological relatedness, genetic homology per se must be determined. Genetic homology is defined in biological terms as the similarity between se59
60
S. G . BRADLEY
quences of loci, but in physical terms, it is the similarity between nucleotide sequences within deoxyribonucleic acid ( DNA) molecules. The genetic potential of an organism is encoded in the linear sequence of the nucleotides in its DNA. These nucleotide sequences determine the sequences of amino acids in structural or enzymic proteins which directly or indirectly constitute the phenotype of the cell. Accordingly, evolutionary divergence from a common ancestor proceeds as the progeny accumulate base substitutions in their DNA. Because of our increased capability to manipulate the DNA molecule, approaches to microbial classification other than traditional determinative systematics can be developed. In fact, an evolutionary approach to bacterial taxonomy, long hindered by lack of an adequate fossil record, now seems feasible. 11.
DNA Nucleotide Composition
The Watson and Crick model of double-stranded DNA predicts that the guanine ( G ) content of a DNA molecuIe equals its cytosine ( C ) content; likewise, the adenine ( A ) and thymine ( T ) contents are also equal. However, the ratio of ( A T ) / ( G C ) , or the mole percent of guaninefcytosine [% G C = ( G + C ) l O O / ( A + T + G + C ) ] may vary from one species to another. As indicated earlier, closely related organisms have very similar nucleotide sequences, and therefore very similar GC ratios. Similarity in % GC does not necessarily indicate genomic similarity but, unlike % GC of DNA from two organisms, establishes that they are not identical. The relationships among selected actinomycetes, with special emphases on streptomycetes, nocardiae, and mycobacteria, have been determined, based upon the nucleotide composition of their DNA (Table I ) . The streptomycetes constituted a homogeneous group whose DNA contained between 69 and 74 mole % guanine cytosine ( % GC). The nocardial and mycobacterial DNA preparations contained 61-69% GC. The nocardial strains fell into either a 62,-64% GC group or a 6749% GC group (22).
+
+
+
Ill. DNA:DNA Association
Because the phenotype of an organism is determined by the nucleotide sequence in its DNA, a comparison of the nucleotide sequences of DNA preparations from two organisms should give a definitive evaluation of their relatedness. Unfortunately, complete direct sequence analysis of a DNA molecule is not currently possible. The complementary nature of the DNA double helix, however, can be used to circumvent these
61 TABLE I NUCLEOTIDE COMPOSITION OF SELECTED ACTINOMYCETE DNA SAMPLES Source
% GCa
Actinomadura Act i n o myces Actinoplanes Ampullariella Dact ylosporangium Micromonospora M ycobacterium M ycococcus Nocardia Oerskovia Planobispora Planomonospora Spirillospora Streptom yces Streptosporangium Streptoverticillium Thermomonospora
68 58 71-73 72 71-73 71-73 64-69 68 61-69 74-76 70-71 72 71-72 69-74 69-7 1 69-7 1 67-69
Values compiled from our published works (4,6,9,14). Q
technical difficulties. It has been established that the two strands of the DNA helix can be separated and specifically reassociated or annealed (19).
The phenomenon of association of complementary strands of DNA provides a powerful tool for exploring the relationships among microorganisms ( 18). Investigators analyzing DNA preparations with 60-70% GC encountered many problems with the earlier hybridization methods (27). Accordingly, my colleagues and I ( 9 , 1 3 ) have modified the method of Warnaar and Cohen ( 2 4 ) for quantitative assay of hybridization between denatured DNA fixed to nitrocellulose membrane filters and free, denatured DNA. We have used this modified technique to assess, on a molecular level, the relationships among representatives of many genera of actinomycetes, in particular Mycobacterium, Nocardia, and Streptomyces (Table 11). The relationships among these organisms are of particular interest because of their special relevance for industry, medicine, and agriculture. Moreover, the taxonomy of the actinomycetes remains a subject for active study and debate. A difficult problem in using nucleic acid association for taxonomic studies is the selection of a system that will give the needed information. There are two general methods to choose between ( a ) association involv-
62
S. G. BRADLEY
TABLE I1 ASSOCIATION OF DNA FIioM Nocardia asteroides STRAINS 300 A N D 330 WITH DNA FROM SICLIK!TEDNOC.\I
