Studies on Induced Variation in the Rhizobia

SCHWINGHAMER, E. A. (Brookhaven National Laboratory, Upton, N. Y.), AND R. L. DALMAS. Studies on induced variation in the rhizobia. II. Radiation sensitivity and induction of antibiotic-resistance markers. Appl. Microbiol. 9:410-414. 1961.-The relation between survival and dose of ultraviolet (UV) light or X rays was, with only one exception, nonlinear for late-log-phase cultures of four species of Rhizobium. The LD90 for different bacterial strains ranged from 400 to 3,000 ergs per mm2 for UV treatment, and from 3.0 to 7.5 kr for X-ray treatment. Minor existing differences in response to some antibacterial compounds (disc tests) were usable as secondary markers for genetic experiments. Spontaneous and radiation-induced mutants, resistant to high levels of several antibiotics, notably dihydrostreptomycin and erythromycin, were isolated for primary marker purposes. A significant phenotypic lag for mutation to dihydrostreptomycin resistance was noted for UV-irradiated cells. The UV-induced frequency exceeded the X-ray-induced frequency by a factor of at least three, at a comparable level of lethality. No significant change in the symbiotic capability was observed in rhizobial strains marked with antibiotics resistance. Attempts to modify infectiveness (noduleforming ability) and effectiveness (ability to fix nitrogen) in the rhizobia are complicated by a number of experimental factors, including airborne or seedborne rhizobial contamination, varying degrees of cross-nodulation, and problems of detecting the rarely occurring variants on the host plant. Experimental evidence for any such modification thus rests primarily on an adequate system of marker characters, rather than on tests of statistical significance. This paper describes the dose-survival response of some rhizobial strains to ultraviolet light (UV) and X rays, and the induction of antibiotics resistance as a marker character. Radiosensitivity determinations and the marking of cultures are being directed toward studies involving modification of the symbiotic characteristics of Rhizobium. MATERIALS AND METHODS Cultures and media. Some of the strains of Rhizobium were provided by the Nitragin Company, Milwaukee, Wisc.; other strains were isolated from field collections of nodules. The nutrient media and nodulation testing techniques used are described in the previous report (Schwinghamer, 1960). The GjSY (glucose-salts-yeast extract) medium and the double layer, surface-layering method of plating were used for the dose-survival experiments and most of the antibiotic experiments, unless otherwise indicated. Radiation treatments. Irradiation factors pertaining to the X-ray unit are the following: G. E. Maxitron 250 therapy unit1; 250 kvp; 30 ma; filter, 1.0 mm Al + 0.25 mm Cu; HVL, 0.9 mm Cu; dose rate in air, ranging from 600 to 700 r per min between experiments. The UV source was a 15-w G.E. germicidal lamp,2 used with a standard fluorescent lamp fixture and voltage regulator. The average 2537 A intensity (calibrated with an Eppley3 thermopile-galvanometer system and a standard lamp) incident at the bacterial suspension surface, 12 in. distant from the lamp surface, was ca. 7.3 ergs per mm2 per sec. This intensity was obtained from a 4-in. slit at the linear center of the foil-covered lamp and allows for approximately 27 % UV absorption by a cellophane membrane (300 PT, uncoated)4 cover placed over the irradiation dish. Cells to be irradiated were generally taken from broth cultures in the late log phase of growth (5 X 108 to 109 cells per ml). The cells were washed by centrifugation and resuspension in a salts solution (GjSY broth without glucose and yeast extract). For mutation experiments involving high-lethality exposures, the titer was increased to the desired level upon resuspension. The chilled cell suspension was exposed in a sterile, cellophane-covered dish assembly and agitated during irradiation by a magnetic stirrer. The same assembly was employed for both UV and X-ray exposure, although the maximal depth of suspension 1 General Electric X-ray Corporation, Milwaukee, Wise. 2 General Electric Cleveland, Ohio 3 The Eppley Laboratory, Inc., Newport Road, R. I. 4E. I. duPont de Nemours and Company, Inc., Wilmington, Del. 410 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom INDUCED VARIATION IN RHIZOBIA for exposure to UV was maintained at 5 mm, as compared to 10 mm for X-ray exposure. Irradiated cell suspensions were chopped for 1 min. in a Waring Blendor to minimize clumping error. In the dose-survival experiments the appropriately diluted cell suspensions were plated in triplicate in a 2-ml soft agar (0.9 %) layer on a 1.5% agar base. Antibiotic experiments. The antibiotic paper-disc (Difco5 Bactosensitivity Disks) test was used for preliminary evaluation of strains to a group of 31 antibacterial compounds. Routine screening experiments of this nature were made to catalog interstrain differences in antibiotics resistance (for marker purposes), and to note the frequency of resistant colonies in inhibition zones as a preliminary indication of mutability in a culture. Antibiotics or drugs subsequently selected for use in isolation of resistant variants were the following: dihydrostreptomycin sulfate, erythromycin,6 penicillin, bacitracin, nitrourantoin.7 The gradient plating technique (Szybalski and Bryson, 1952) was employed to determine the approximate concentration threshold for inhibition, as well as 5 Difco Laboratories, Inc., Detroit, Mich. 6 Erythrocin, Abbott Laboratories, North Chicago, Illinois and Ilotycin, Eli Lilly and Co., Indianapolis, Ind. 7Furadantin, Eaton Laboratories, Norwich, N. Y.