APPLIED MICROBIOLOGY, Dec. 1971, p. 1076-1084 Vol. 22, No. 6 Copyright © 1971 American Society for Microbiology Printed in U.S.A. Physiological Studies of an Oligosporogenous Strain of Bacillus popilliaet RALPH N. COSTILOW AND WILSON H. COULTER2 Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48823 Received for publication 6 August 1971 A relatively small but consistent increase in the frequency of spore formation by an oligosporogenous strain of Bacillus popilliae (NRRL B-2309M) was obtained by adding 0.1% sodium pyruvate to the sporulation medium. The frequency of spore formation was essentially the same when a low level of glucose, trehalose, or glu- cose-6-phosphate or a high level of a-methyl-D-mannoside was added as the carbon and energy source. Many other variations in the cultural medium and cultural con- ditions failed to enhance spore formation of 2309M, and no spores were found in four asporogenic strains under any of the conditions tried. There were no significant differences between the 2309M strain and three nonsporeforming cultures with re- spect to (i) the rate and extent of growth, (ii) the rates of glucose utilization, or (iii) volatile acid production and utilization. None of the cultures tested was found to produce detectable levels of extracellular protease or an antibiotic. The only con- sistent marker found associated with spore formation was the development of cata- lase activity, and this activity was stimulated by heating at 80 C for 10 min. This was not found unless morphological evidence of spore formation was observed. The germination of the spores formed by 2309M in vitro was stimulated by heat shock and by the addition of pyruvate to the germination medium. The isolation of a variant culture of Bacillus (22) with asporogenic strains ofB. popilliae. Stud- popilliae (NRRL B-2309M) which sporulated at ies involved the influence of various energy significant frequencies in vitro (22) provided the sources on sporulation; comparisons of growth opportunity to compare some of its physiological rates, glucose utilization, pH changes, and volatile properties during spore formation with the parent acid production; and the production of sporula- and other strains which did not produce spores tion-specific enzymes and antibiotics during under the same conditions. It was hoped that such colonial development on the sporulation medium. studies would provide information which could Also, some observations of the germination of be used in devising conditions for sporulating the spores produced in vitro were made. wild-type cWltures in vitro. McKay et al. (10) demonstrated that oligosporogenous variants all MATERIALS AND METHODS are able to oxidize acetate, whereas cultures ini- Cultures and cultural methods. All parent cultures from of the do not. of B. popilliae used were obtained from Northern Uti- tiated spores wild-type strain lization Research and Development Division, Agri- However, other asporogenic variants oxidize ace- cultural Research Service, Peoria, Ill. Also, spores of tate even more rapidly than the strains producing NRRL B-2309 produced by injection of Japanese spores. Therefore, although the derepression of beetle larvae were supplied by Grant St. Julian, Jr., of acetate oxidation may be an important factor, it the above laboratory. The oligosporogenous strain obviously does not constitute the only difference used was NRRL B-2309M (22), and the asporogenous between strains sporulating in vitro and those strains were NRRL B-2309S, B-2309PA, B-2309N, which do not. and B-2309MC. These strains will be referred to in This report covers the results of many experi- this paper as 2309M, 2309S, etc. The history of the ments conducted to the strains used and procedures used for their maintenance compare physiological have been described (10). properties of the sporogenic strain of Sharpe et al. Growth and sporulation. The basic sporulation me- was ' Journal article no. 5553, Michigan Agricultural Experiment dium that described by Sharpe et al. (22) except Station. that the 0.05% trehalose was replaced by 0.05% glu- IPresent address: Department of Biology, University of New cose. The medium contained 1% Mueller Hinton Brunswick, Fredricton, N.B., Canada. broth medium solids (Difco), 1% yeast extract (Dif- 1076 VOL. 22, 1971 OLIGOSPOROGENOUS STRAIN OF B. POPILLIAE 1077 co), 0.3% K2HPO4, and 0.05% glucose and will be pounds present was estimated by determining the referred to as MYPG. Pyruvate (0.1%) was added to radioactivity remaining after acidification to below pH this medium where indicated. The substitution of glu- 6.0 to liberate dissolved CO2 and subtracting the values cose for trehalose was made after we found that it did obtained for volatile acid. The radioactivity in the not influence the sporulation frequency which we aqueous samples was counted in the scintillation solu- achieved. All of the components of the medium except tion of Bray (1). A 1-ml sample of the acidified mixture the agar were filter-sterilized. was placed in the outer well of a Conway dish along Spores used for inoculating plates were harvested with 1 ml of concentrated H2S04. Hydroxide of hya- from plates by washing with three 5-ml volumes of mine (1 ml) was placed in the center well. After 48 hr water and were routinely heat-shocked at 60 C for 10 of incubation, the hydroxide of hyamine was quanti- min. In a few instances (see below), the vegetative cells tatively removed with methanol and made to a total in spore-containing colonies were destroyed by invert- volume of 2 ml. Portions of this solution were placed ing the petri dish over a piece of chloroform-soaked in a toluene-basedscintillation fluid (11), andthe radio- cotton for 5 min prior to harvesting, and no heat shock activity was counted. These counts were considered was used. The plates were spot-inoculated to obtain to represent the volatile acid. A Nuclear-Chicago Mark 10 evenly spaced colonies per plate. An inoculator was I scintillation spectrometer was used for all I4C deter- fashioned from a no. 13 rubber stopper with ordinary minations. straight pins stuck into it. After sterilization, the pin Catalase production. Cells and spores were har- heads were dipped into a suspension ofspores and then vested from the sporulation medium at various inter- touched to the agar surface. Inocula for the asporo- vals as described above. They were assayed for catalase genic strains were produced in the Trypticase-yeast activity by using the manometric procedure of Law- extract-glucose (TYG) medium described previously rence and Halvorson (8) except that the oxygen release (12). during the first 4 min after adding substrate was used The frequency of sporulation [(number of spores/ to calculate activity. Cell extracts were prepared by total number of cells and spores) X 100] was deter- exposure to ultrasonic oscillation for 20 min in a 100-w mined by direct counting of the number of character- ultrasonic disintegrator (Measuring and Scientific istic spores with parasporal bodies present in a popu- Equipment, Ltd., London). Proteins were estimated lation of at least 200 cells. The suspensions counted on 1 N NaOH extracts (40 C for 2 hr) of hot (90 C for were washed from single plates as described above. For 30 min) 5% trichloroacetic acid extracts of cells by the estimates of growth by optical density at 620 nm, the method of Lowry et al. (9). Protein in cell extracts was suspensions were washed twice with 0.01 M potassium estimated by this method without previous extraction. phosphate (pH 7.4) and resuspended in a total volume Dry weights of cells and spores were determined after of 25 ml of the same buffer. A Gilford model 2000 drying at 110 C for 24 hr. spectrophotometer was used for optical density meas- The refractile bodies used were produced as de- urements. scribed previously (12). The spores from Japanese Analytical measurements. Plates containing the beetle larvae were washed in 0.01 M phosphate buffer MYPG plus pyruvate medium to be used for analysis (pH 7.4) six times before testing for catalase. of changes occurring during colonial development and sporulation were weighed immediately after they were RESULTS poured and cooled. They were dried for 2 to 3 days and inoculated as described above. One plate was Effect of carbon and energy sources on sporula- used at each interval during incubation starting at zero tion. During the course of these investigations, a time. The cells were wiped from the surface with non- number of different carbon and energy sources absorbent (delicatessen) paper, and the plate was were tested for their influence on the frequency of weighed to determine the loss due to evaporation. The sporulation of strain 2309M (Table 1). It should agar was cut into strips, and macerated by forcing it be noted here that the data given represent the through a 25-ml syringe without a needle. The syringe of the percentage of the was rinsed with water equivalent to two volumes of microscopic estimate the original weight of the medium and combined in a population represented by those sporangia con- 250-ml flask with the macerated medium plus enough taining a characteristic spore and parasporal water to correct for the weight loss during incubation. body. We routinely observed in the spore-con- The stoppered flasks were incubated for 2 hr at 30 C taining cultures an equal or higher percentage of on a rotary shaker to allow for equilibration and other forms which were typical of the refractile allowed to sediment; 25 ml of the aqueous supernatant bodies described previously (12). The addition of solution was clarified by centrifugation at 12,000 X g 0.1% sodium pyruvate to the glucose-containing for 10 min. medium had the most pronounced effect on the The pH of this extract was read directly.