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.
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.
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.
The theory that the Mycoplasmataceae (pleuropneumonialike organisms) are naturally occurring L-forms of bacteria is based on the similarities between the Mycoplasmataceae and L-forms of bacteria derived in vitro.1 Interest in this hypothesis has been stimulated by recent reports of the occurrence of L-forms in vivo2-4 and the lack of categorical morphologic' and physiologic5 differences between Lforms and the Mycoplasmataceae. Criteria established to confirm or exclude a Mycoplasma species as the L-form of a bacterium must be applicable to the relationship of an L-form to its known bacterial parent. Therefore, the presence or ab-
The colonial morphology of some strains of Pseudomonas pseudomallei was correlated with certain biochemical and physiological traits. After 3 days of growth on Wahba or heart infusion agars, smooth-colony strains generated toxic amounts of ammonia. Under the same conditions, the rough strains simultaneously produced oxalic acid which decreased the inhibitory concentration of ammonia. The ammonia-ammonium concentrations in smooth cultures exhibited certain bacteriocin-like characteristics. An unusually stable, smooth strain (strain 165) was chosen to compare and emphasize any differences with typical, rough strain 7815. Three-day-old smooth cultures grown on Wahba agar containing 3% (w/v) glycerol demonstrated ammonia toxicity. The substitution of glucose for glycerol completely obviated this toxicity. In highly aerated Wahba broth containing glucose, the amount of ammonia found in strain 165 smooth cultures and the amount of oxalic acid found in strain 7815 rough cultures were greatly reduced. In Difco nitrate broth smooth strain 165 did not form gas, and it reduced nitrate to nitrite only. Strain 7815 produced a gas and reduced both nitrate and nitrite. Antibiosis among bacterial strains is a common occurrence and is sometimes useful as an aid in laboratory identification. One of the most striking antagonisms in this respect is attributed to a class of compounds called bacteriocins. The bacteriocins are generally defined as protein-like, naturally occurring bacterial antibiotics which act principally on strains of the same species as the producer or on genetically related species. Some bacteriocins act on species of other genera (4, 25). Our efforts to find bacteriocins among strains of Pseudomonas pseudomallei proved fruitless. However, another type of antagonism was found which resembled bacteriocin antibiosis, but was essentially due to ammonia excretion. This paper describes the conditions under which ammonia toxicity was observed and some interesting correlations among colonial morphology, physiological and genetic traits. MATERIALS AND METHODS Strains and cultural conditions. Eighteen isolates of P. pseudomallei were obtained from water, soil, and animal sources in Vietnam, Malaysia, and Thailand. P. mallei (synonym Actinobacillus mallei, strains 4 and 3873) and P. cepacia (synonym P. multivorans, strains 17616 and 17759) were described previously (26). P. aeruginosa pyocin indicator strains were obtained from R. J. Zabransky (38). Strains were maintained on Difco brain heart infusion agar containing 3% (w/v) glycerol (glycerol agar). Survey for inhibiting strains. The survey procedures were essentially those of Darrell and Wahba (6), except that the basal medium (36) was heart infusion broth (Difco) containing 10-6 M iodoacetic acid, 0.1% sodium citrate, 0.1% K2HPO4, 0.0018% phenol red dye, and 2% agar (Wahba agar). In the survey, a candidate inhibitor strain was streaked on Wahba agar and incubated at 37 C for 3 days. The cultures were then exposed to CHClI vapors for 30 min, the growth was scraped off w...
357Summary Klebsiella pneumoniae was isolated from lesions in 2 dead and 82 ill animals in a breeding colony of 2300 Wistar rats. The clinical signs were unilaterial and bilateral fluctuating masses in the cervical and inguinal areas, and focal cutaneous ulcers in the ventral neck. Cervical and inguinal lymphadenitis with abscess formation were found on microscopic examination. Lesions also occurred in visceral organs. Although characteristic of the natural infection in most species, no respiratory lesions were seen in this epizootic episode. A capsular serotype 5 K. pneumoniae which did not utilize malonate was the only bacterial strain cultured from the lesions, but other K. pneumoniae strains that utilized malonate and were untypable hy capsular serology were cultured from throats and faeces. 30% (6/20) of asymptomatic animals tested had both types of K. pneumoniae in their faeces.
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