No abstract

The deoxyribonucleic acids (DNA) from phenotypically unusual Leptospira strains were compared to each other and to reference strains of existing genetic groups. In the "pathogenic" genetic complex, three groups emerged as genetically distinct. Representative examples of these groups were bataviae strain Van Tienen, javanica strain Veldrat Bataviae, and ranarum strain Iowa City Frog. The serologically different muenchen strain Muenchen 90C, icterohaemorrhagiae strain RGA, and kabura strain Kabura were found to be closely related to bataviae strain Van Tienen. The "saprophytic"genetic complex also contained three inter-related groups. The representative examples were patoc strain Patoc I, codice strain CDC, and Turtle strain A-183. Strain 3055 of serotype illini appeared t o have unique nucleotide sequences and was placed in a third genetic complex of its own. Partial relatedness between DNA could be emphasized by increasing the salt concentrations in the incubation media. This could not be attributed t o the methods of annealing but appeared to be dependent upon the genetic relatedness between the heterologous DNA.The genus Leptospira can be separated into two major divisions, the pathogenic and the so-called "sap rop hy t icy' or "bifle xa " lep t 0-spiras. The latter are usually found in fresh surface waters and are rarely found in animals. In addition to infectivity, various phenotypic properties serve to separate members of the two divisions (1 8). Using deoxyribonucleic acid (DNA) base composition determinations and specific DNA-DNA annealing tests in agar matrices, Haapala et al. (7) demonstrated genetic differences between, as well as within, serotypes in the two divisions. Two distinct genetic groups were demonstrated among selected strains within each of the pathogenic and biflexa serotypes. The serological relationships among lep tospiras did not necessarily denote genetic relatedness; however, strains of the same or very similar serotypes appeared to be in the same genetic group. The two genetic groups of pathogenic lep tospiras could be differentiated from each other and from the two biflexa groups by a number of phenotypic characteristics which also served as a basis for the leptospiral groups described by Johnson and Harris (9). The principal differentiating attributes were lipase production and relative resistance to the bacteriostatic action of 8-azaguanine (AZA) and 2,6-diaminopurine (DAP).The genetic groups of pathogenic strains represented by strains of serotypes bataviae and javanica corresponded to the Johnson and Harris biological groups 1 and 2, respectively. Group 1 strains were sensitive to AZA and DAP and had lipase activity, whereas group 2 strains were sensitive to AZA, resistant t o DAP, and lacked lipase activity. The two genetic groups of biflexa strains were phenotypically indistinguishable and fit into the Johnson and Harris biological group 3 , which was characterized by resistance to both purine analogues and by positive lipase activity.

Annealing experiments on membrane filters were carried out with deoxyribonucleic acids (DNA) from selected strains of the nomen-species of Pseudomonas, Actinobacillus, Chromobacteriwn, and Micrococcus, with the use of DNA of Pseudomonas pseudomallei and Actinobacillus mallei as reference materials. Under the usual conditions employed in these experiments, the results were not quantitatively reproducible. Incorporation of dimethylsulfoxide (DMSO) into the incubation medium greatly increased differences in comparative binding. DNA binding in agar matrices was examined in the presence and absence of DMSO at various incubation temperatures. It was found that the greatest specificity, stability, and total binding for DNA containing high amounts of guanine and cytosine occurred in the presence of DMSO. Under the most stringent annealing conditions permitted in agar, DNA species from P. pseudomallei and A. mallei in the presence of DMSO demonstrated interspecific relative bindings of 76 to 86% when compared to the homologous reactions. The thermal elution midpoints (Em) of these duplexed interspecific DNA species were quite close to the homologous Em values. The relative bindings of P. multivorans DNA types to either reference DNA ranged between 6 to 27%, and the Em values were 4 to 7 C less than those for the homologous reactions. were extracted from these organisms and characterized by composition and homologous and heterologous annealing abilities.

Four distinct genetic groups of leptospiras were demonstrated among selected pathogenic and "biflexa" serological types. Pathogenic leptospiras could be divided into two groups on the basis of per cent guanine + cytosine (GC) in their deoxyribonucleic acid (DNA). One group had 36 1%, the other 39 4 1%. The biflexa strains had DNA of 39 + 1 % GC, but were further separated into two groups on the basis of DNA-annealing tests. Strains within groups had a high degree of specific duplex formation (75 % binding or more with reference to the homologous DNA). There was little or no genetic relatedness between strains of the four groups (less than 10% DNA homology). The thermal elution midpoint of heterologous DNA duplexes was always lower than the homologous reaction. The serological relationships among strains were not meaningful in terms of relatedness determined by specific duplex formation.

acid homologies of selected bacteria, L forms, and Mycoplasma species. J. Bacteriol. 90:1200-1204. 1965.-The molar per cent of guanine plus cytosine (G + C) in the deoxyribonucleic acids (DNA) of Proteus mirabilis, strain 9, and its stable L form was determined by thermal denaturation and found to be approximately 39.5%O G + C. The DNA homologies of this bacterium and its L form were estimated by the agar-column technique and were equivalent in their abilities to anneal and form specific duplexes. The next series of comparisons were performed between two Mycoplasma species and their often suggested bacterial parent. The G + C ratios of M. gallisepticun (32.7%o), 31. gallinarum (28.1%c), and Haemophilus gallinarum (41.9%) varied to a high degree. In the homologous system, the denatured DNA of H. gallinarum trapped in agar bound approximately 40%O of its sheared, denatured, and H'-labeled DNA. In comparison, the nucleic acids of M. gallinarum and M. gallisepticum were incapable of binding the labeled DNA of H. gallinarum. These findings provided evidence that the two strains of M1ycoplasma were not derived from H. gallinarum.