Thermal Stability of DNA: DNA Hybrids within the Genus Agrobacterium

Abstract

S U M M A R YMolecular hybrids were prepared between unlabelled DNA from representative strains of eleven genetic races of Agrobacterium and [I4C]DNA from typical strains of each of the three main races. The thermal stability of each hybrid was determined. The nature of the hybrids formed varied with the incubation temperature and the kind of DNA used. Hybridization in 2 x SSC-30 % dimethylsulphoxide below 59 "C yielded two kinds of hybrids: a labile one of unknown nature, denaturing below 59 "C, and a more or less stable hybrid denaturing above that temperature. The latter was the only one formed in hybridizations at or above 59 "C. There were three kinds of stable hybrids. Within each of the main Agrobacterium races thermal stability of the molecular hybrid was about the same (within 2 "C) as for the homoduplex. Between two races of 50 % DNA relatedness, the duplexes were about 6 "C less stable. Between races of 10 to I 5 % DNA relatedness, the duplexes were weak, and the stability was at least 13 "C lower. The stability of the hybrids decreased concomitantly with the degree of DNA relatedness. The decreased hybrid denaturation curve is not due to AT-rich sequences. The less two races of agrobacteria appeared to be evolutionarily related, the more mutations occurred within the common part. I N T R O D U C T I O NFrom previous studies it appeared that the genus Agrobacterium is genetically very heterogeneous. DNA of cluster I (typical Agrobacterium tumefaciens and A. radiobacter strains), cluster 2 ( A . rhizogenes and atypical A . tumefaciens strains), the 'rubi' group and two very small groups hybridize at about 10 to 15 %. Within cluster I the DNA of the seven groups hybridize at about 50 % and within each group at least 80 % ( Mutational events modified considerable parts of the genomes, preventing in vitro molecular hybridization. Considering the evolutionary history of a bacterial genus, one can wonder whether mutations also occurred within the common DNA parts. For an experimental answer to that question we prepared a number of DNA-hybrids between the genomes of different Agrobacterium groups and clusters, and determined their thermal stability which is interpreted as a measure of base-pairing imperfections, and mutational differences (Brenner & Cowie, 1967). We established that decreased hybrid denaturation curves were not due to preferential binding of heterologous AT-rich DNA sequences.

Numerical Taxonomic Analysis of Agrobacterium

“…The groupings obtained with numerical analysis agree on the whole excellently with the results from DNA: DNA hybridizations, DNA hybrid stability (De Ley, Tijtgat, De Smedt & Michiels, 1973) and protein electrophoresis (J. De Ley & K. Kersters, unpublished).…”

Intra- and Intergeneric Similarities of Agrobacterium Ribosomal Ribonucleic Acid Cistrons

“…When more strains are used, the shape and size of the area occupied by a genus is a measure of its heterogeneity. From previous research in this laboratory (16,17,25,42) we know that Agrobacterium is a rather,heterogeneous genus, consisting of two large genetic clusters, 1 (exemplified by Agrobacterium radiobacter and A. tumefaciens strains, numbers 1 to 15) and 2 (exemplified by the rhizogenic and atypical A. tumefaciens strains, numbers 16 to 19), a small Agrobacterium rubi cluster (numbers 20 and 21), and a few isolated strains (numbers 22,23, and 24). These clusters hybridize at about 15% DNA homology among each other.…”

Cytochromes c‐556 from Three Genetic Races of Agrobacterium

“…Although the sequence information on B6 cytochrome c-556 is limited at present to the first eight residues, the complete identity of the N-terminal region of cytochrome c-556 from strains B2a and B6 strongly suggests that the latter cytochrome is likewise a member of class TI. This is not surprising since B6 and B2a are strains of the same genetic race with genome DNA of nearly 100 % similarity [1,2]. Although both cytochromes have the same isoelectric point, it is unlikely that the sequences are completely identical because there are slight differences in the amino acid compositions (Table 4).…”

“…The races B6 and T T I I~ are members of the so-called Agrobuctc.rium 'cluster 1 ', those of race rhizogenes constitute 'cluster 2' [4]. The genome-DNA from strains of race B6 has 50 % similarity to the genome-DNA from strains of race TTI11, but each of these DNA types has only 10 to 15 similarity to the DNA of strains of the rhizogenes race [1,2].…”