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 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. Pyruvate of formation of those tested. was assayed by the procedure of Friedemann and frequency spore Haugen (4), and the colorimetric assay outlined in However, it was not effective when used alone. A Neish (14)-as used to detect lactate. Glucose utiliza- low level ofglucose-6-phosphate supported sporu- tion was estimated by the reduction in total reducing lation as well as glucose, but higher levels progres- sugars present. In one experiment, [U-14C]glucose sively reduced the frequency of sporulation. In (7,900 counts per min per- Amole) was added to the contrast, with a-methyl-D-mannoside, the fre- medium, and the decrease in the nonvolatile com- quency of sporulation increased as the concentra- 1078 COSTILOW AND COULTER APPL. MICROBIOL.

TABLE 1. Effect of various carbon and energy development; (v) fractionation of yeast extract sources on sporulation into anionic and cationic components by the use of Dowex 50, Cl--form and H+-form columns, Per cent sporesb respectively, and using the concentrates to replace Compound(s) addeda the yeast extract in the medium and as supple- ControlC Treatedd ments to it; (vi) the addition of reduced glutathi- one (0.01 %) initially and, also, by feeding as in None...... 2-6 <1 (iii) and (iv); (vii) the addition of 5% sterile defi- ...... 2-6 2-6 Trehalose (0.1%) blood to the agar before autoclaving or Trehalose (0.05%) ...... 2-6 2-6 brinated Glucose (0.05) by adding various levels of hemin before filtration + Pyruvate (0.05%) ...... 3 11 of the nutrient components; (viii) addition of ± Pyruvate (0.1%) 12 0.04% dipicolinic acid; and (ix) addition of a + Pyruvate (0.2%) 12 variety of metal ions to the regular medium and + Lactate (0.05%) ...... 3 3 to the medium prepared with a solution of Muel- + Gluconate (0.05%) 3 2 ler Hinton solids and yeast extract which had been Pyruvate (0.05%) ...... 3 1 treated with Dowex 50, Na+-form resin at pH 6.0 Glucose-6-phosphate to remove divalent cations. A number of similar (0.1%) ...... 5 7 (0.25%).. 3 experiments were performed with this medium and (0.5%) ..1 the yeast extract-peptone medium of Pheil and a-Methyl-D-mannoside Ordal (16) in broth cultures incubated on a shaker (0.05%) ...... 5 1 without success. Strains of B. popilliae which do (0.2%)... 2 not produce spores under the conditions outlined (0.5%) ...... 7 by Sharpe et al. (22) were also tested for spore (1.0%) ..2 formation in all of the above experiments without success. When 0.2 to 0.5% salicin was used as the Compounds indicated were added to the of Mueller Hinton-yeast extract-phosphate medium. energy source in a broth medium (16), many bPer cent sporulation was estimated micro- the cells of most strains of B. popilliae tested ap- scopically after a minimum of 21 days of incuba- peared to be swollen in a manner characteristic of tion. sporangia. However, no other evidence of spore c Control medium (MYPG) contained 0.05% formation was noted. glucose. Growth and glucose utilization during colonial d Medium contained the carbon sources indi- development. Figure 1 compares the growth rates cated. and rates of glucose utilization of the oligospo- rogenous strain (2309M) with the ranges observed tion was increased up to 0.5% but decreased at with three nonsporulating strains. There was a the 1% level. Growth on the a-methyl-D-man- delay of 12 to 18 hr in the initiation of rapid noside medium also increased as the concentra- growth of 2309M as compared to the other three. tion of sugar increased. The dry weight of cells in This was expected since a spore inoculum was the 10 colonies from one plate of the media con- used with 2309M and a cell inoculum with the taining 0.05, 0.2, and 0.5% of this sugar averaged other strains. The mean generation time during 2.4, 5.5, and 8.3 mg (dry weight), respectively. exponential growth for all strains was estimated This was after 20 days of incubation. at about 3.75 hr, which is considerably longer Other attempts were made to increase the fre- than that observed in broth cultures (2.5 to 3.0 quency of sporulation in this medium but without hr). As in other aerobic , spore formation significant success. These included (i) addition of occurred in 2309M as the culture approached sta- other compounds as carbon and energy sources tionary phase of growth. An occasional swollen including fructose, sucrose, a-methyl-D-glucose, cell was observed after 4.3 days of incubation, and salicin, soluble starch, and cellobiose; (ii) prepara- the maximum percentage of spores (6%) was ob- tion of MYPG plus pyruvate medium by using served after 5.1 days of incubation. The period the supernatant solution of centrifuged stationary- between exponential growth and the stage of max- phase broth culture (spent medium) and adding imum growth was prolonged (-3 days). This various carbon and energy sources; (iii) feeding of prolonged period of a declining growth rate was glucose by adding a drop of sterile 0.2% glucose correlated with a constantly decreasing rate of to a sterile filter paper disc on the agar surface at glucose utilization. There was essentially no fur- dawily intervals; (iv) adding 10-3 M adenosine ther increase in cell mass after all of the glucose -3',5'-monophosphate (cyclic AMP) initially and was exhausted from the medium. The utilization of also by adding a drop of 102 M to a filter paper the added pyruvate paralled that of glucose. disc as in (ii) at various intervals during colonial Changes in pH and the production ofvolatile acid VOL. 22, 1971 OLIGOSPOROGENOUS STRAIN OF B. POPILLIAE 1079

.~I FIrST SPORES 0.50 8.0

7.8 0.10o - ",; 2 .0

C: ;14 :;L 0.05 0.50 E 7.6

t:hE -j 7.4 Q. 0.005 0.1 0 0.005 -I1 0.05 E 7.2 Il I E 10.01 ( 0wS.( 2 4 6 8 10 12 W) DAYS 0 FIG. 1. Comparison ofgrowth andglucose utilization during colonial development on MYPG plus pyruvate medium of an oligosporogenous strain (2309M) with that of three nonsporulating strains ofB. popilliae. The optical density at 620 nm was taken of the washed cells from 10 colonies on a plate suspended in 25 ml of 0.01 M potassium phosphate buffer. Solid lines represent growth and the dashed lines, glucose utilization. Sym- bols: 0, *, 2309M; I and T, ranges for 2309PA, 2309S, and 2309N. 2 4 6 8 10 12 14 16 18 20 during colonial development. There was not a suf- DAYS ficient amount ofvolatile acid accumulated during FIG. 2. Comparison ofpH changes and the produc- growth to estimate by steam distillation. There- tion of [14C]volatile acid from [U-'4C]glucose during fore, [U-'4C]glucose was added to the MYPG colonial development ofan oligosporogenous strain and plus pyruvate medium, and the volatile acid was a nonsporulating strain of B. popilliae. Symbols: 0, estimated by radioactivity measurements. It is [14C]nonvolatile residue; , [14C]volatile acid; i, pH. realized that there may be a significant loss of vol- atile acid during incubation of the plates, but the Spore formation was observed with 2309M (see data should reflect a general pattern. All four inset in Fig. 2) after the pH of the medium had strains listed in Fig. 1 were tested, but the results started to increase. with only two ofthese will be presented since they Development of sporulation-specific antibiotics are representative. There was nothing unique about and enzymes. All attempts to demonstrate the pres- the pH and volatile acid patterns of the oligo- ence of an antibiotic during sporulation of strain sporogenous strain (Fig. 2). The pH values de- 2309M failed. Staphylococcus aureus, Escherichia clined to a minimum of 7.2 to 7.3 during 4 days of coli and Bacillus megaterium were all used as test incubation and then increased dramatically. Max- organisms. Dilute suspensions of the test organ- imum volatile acid levels were observed at 4 days ism (,106/ml) were streaked adjacent to colonies also, but the acid levels declined only slightly of B. popilliae at various stages during colonial thereafter. The total volatile acid accumulated in development and spore formation. In no instance the asporogenic (2309S) strain was about two was any evidence ofinhibition noted. Similarly, no times higher than that in 2309M but still repre- intracellular or extracellular protease activity was sented only about 46% of the nonvolatile '4C detectable with 2309M at various stages of colo- which had disappeared from the medium. The nial development. Tests were conducted by (i) nonvolatile 14C disappeared at a slower rate from adding 1% casein to the sporulation medium and 2309M than from 2309S, and the maximum vola- observing for zones of hydrolysis around the colo- tile acid observed amounted to only 36% of the nies, (ii) incubating colonies and the surrounding nonvolatile '4C which had disappeared. Tests for excised medium or cell extracts with azo-albumin the possible intermediate accumulation of lactate and measuring the release of trichloroacetic acid- in these cultures were negative. The results with soluble chromophoric groups (2), and (iii) meas- strain 2309N were essentially identical to 2309M, uring the release of 5% trichloroacetic acid-solu- and those for 2309PA were very similar to 2309S. ble '4C from [14C]protein during incubation with 1080 COSTILOW AND COULTER APPL. MICROBIOL. TABLE 2. Development of catalase activity durinig Incubation of 0.2 ml of the sameB. cereus protease sporulation of Bacillus popilliae (2309M) with 1 ml of azo-albumin solution for 30 min re- sulted in an increase in the optical density at 340 02 from H202 [pliters per min nm of 0.13. Time of per mg (dry The only enzyme activity which was consist- incubationa Microscopic observation weignt)] (day) ently associated with sporulation was catalase. Previous data have demonstrated that vegetative heated Heated5 cells are devoid of this enzyme (15, 25) but that spores produced in vivo and refractile bodies pro- 3 All cells 0 0 duced in the laboratory have significant catalase 4 Few swollen cells 0 0 activities (12). Catalase was also produced by 5 Few swollen cells 0 0 strain 2309M during sporulation in colonies 6 Few spores (<1%) 1.7 0.8 (Table 2). It is obvious that catalase activity ap- 7 2-4% Spores 1.1 1.0 10 2-4% Spores 1.5 1.5 peared during sporulation, and that it was com- 17 2-4% Spores 1.9 1.9 pletely resistant to heating at 80 C for 10 min. 26 Cells separated from 2.2 2.2 However, the data indicate that it was produced spores by centrif- early in the sporulation process. Thus, the cells ugationc separated from the spores by centrifugation were Spores (-80%) after 3.6 4.7 about 50% as active as the fraction greatly en- separation from riched in spores. No evidence of significant cata- cellsc lase activity was detected with any of the asporo- genic strains during colonial growth on the same a Cells were harvested from plates at the time indicated and washed one time; catalase was de- medium. Two of these strains (2309MC and termined manometrically. Reaction mixture con- 2309N) were derived from 2309M. tained 0.5 ml of 0.1 M phosphate buffer (pH 7.0), Comparisons of the catalase activity in the cells approximately 100 mm H202, 5 to 10 mg (dry and spores of strain 2309M produced in vitro weight) of cells, and water to 3 ml. with that of refractile bodies produced as de- b 80 C for 10 min. scribed previously (12) and with spores from c Nonsporulated cells were separated by cen- Japanese beetle larvae are shown in Fig. 3. Al- trifuging the suspension for 5 min at 3,000 X g. though the total activity of the refractile bodies The cells in the supernatant fluid were then cen- was high, it was mostly heat-labile. In contrast, trifuged at 10,000 X g for 15 min and resuspended. There were no refractile spores evident in this the activity associated with the cells and spores suspension. The spores were concentrated by re- harvested from colonies on plates was stimulated suspending the pellet of the 3,000 X g centrifuging by heating at 80 C for 10 min. The partial heat in 10 ml of water and repeating the process five lability of the enzyme observed with the spores times. Each time, the top layer of the pellet which from larvae may have resulted from contamina- was largely cells was discarded. The percentage of tion with the heat-labile catalase from larval spores in the final suspension was estimated by hemolymph or catalase-producing . Al- microscopic examination. though the spores were washed six times before testing, there is no assurance that they were free cells from colonies or with cell extracts. The latter of hemolymph. Also, it is not possible to prevent protein was prepared from cells of B. popilliae some degree of microbial contamination of the grown in 250 ml ofTYG broth to which 25 ACi of a spores when harvesting from larvae. The catalase t U-14C] amino acid mixture was added. The radio- associatedwiththe cells, spores, and refractile bod- active protein was prepared as described by Sadoff ies was destroyed by autoclaving. However, in et al. (20), and the final solution contained about 1 some experiments, a low background of up to mg of protein per ml with a specific activity of 1.5 10% of the total activity was noted after auto- x 105 counts per min per mg. Incubation of 50 claving. Also, some sensitivity (up to 50%) of ,uliters of this ['4C]protein with 10 ,lAiters of pro- catalase to heating at 80 C for 10 min has been tease from B. cereus (20) in a total volume of 0.1 observed with cells and spores of2309M produced ml for 30 min at room temperature resulted in the in vitro after storage for several weeks at -20 C. release of sufficient trichloroacetic acid-soluble As evident in Table 3, the catalase activity in material to give a count of 186 counts/min in a the spores freed of cells, sporangia, and para- sample equal to 10 ,uliters of the original reaction sporal bodies appeared higher than observed with mixture. This is equal to about 19% of the total an enriched intact spore suspension noted earlier radioactivity. When water was substituted for the in Table 2. Unfortunately, the spore yields were so protease, an equivalent trichloroacetic acid-solu- low we did not have a sufficient quantity to per- ble sample contained only 14 counts/min of '4C. form other experiments with them. The cell ex- VOL. 22, 1971 OLIGOSPOROGENOUS STRAIN OF B. POPILLIAE 1081

REFRACTILE BODIES SPORES FROM LARVAE IN VITRO CELLS and SPORES 80I Control 0---0-_ Heated, 80C- 10min A- -- Autoclaved CZ *~0 601 ol z Ar 401 9, -,1 .- -o 20 .0 r . .1 /r

I AJ^^L_MWAA-_AL-----JL.J- -A-A_ A- -^llI I u A IAL- -I--I -,,L-md -A -1I m CA-1^- II-NEII&M -|A__- A-L _ -Xm WW%8f&l Li Li A, W' 8-ANk- -,&- &A &A &A && 5.3 6.6 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25 MINUTES FIG. 3. Catalase in refractile bodies, spores from larvae, and in cells and spores produced in vitro.

tract was fractionated, and the highest catalase TABLE 3. Catalase activity of spores and of various activities were found in the supernatant solution fractions of cells of Bacillus popilliae 2309M after centrifugation at 20,000 X g (Table 3). The 02(pliters per min per mg of activity was not decreased by prolonged dialysis or protein) by precipitation with (NH4)2SO4.The rate of 02 Fraction released from H202 increased in a linear manner 10 mm Control 10 mm KCN with protein concentration with the dialyzed ex- NaNs tract. Also, the enzyme was inactivated by perox- ide by a first-order reaction. The activities in all Cells and spores. 6.6-8.1 3.0 4.2 Sporesa ...... 30.0 cell fractions were strongly inhibited by azide and Cell extract...... 4.1 cyanide except for that in the 20,000 X g pellet. 2,000 X g super- Thus, it is likely that the activity is primarily due natant fluid...... 4.1-5.5 1.4 1.6 to a heme-type catalase. 20,000 X g super- Germination of in vitro spores. During the iso- natant fluid...... 4.4-5.5 0.6 1.1 lation of oligosporogenous mutants of B. popil- 20,000 X g pellet ... 2.7-3.1 1.9 2.0 liae, it was observed that colonies developed from Dialyzed 20,000 X g heat-shocked (60 C for 15 min) samples on plates supernatant fluid. 6.0 0.7 1.2 of MYPG plus pyruvate but not on the same me- (NH4)2SO4 precipi- dium without pyruvate (Bhumiratana and Cos- tatec ...... 7.6 tilow, unpublished data). Plating of heat-shocked a Cells and spores were exposed to ultrasonic and chloroform-treated suspensions of in vitro oscillation for 2 min which was sufficient to break spores produced on media with and without cells and to remove sporangia and parasporal pyruvate confirmed these observations (Table 4). bodies but did not break the spores. The pellet of The numbers of colony-forming units observed the 2,000 X g centrifugation contained the spores. with the pyruvate-containing media were from 2 The spores were washed three times with water to to over 100 times greater than the controls. remove debris before testing. Pyruvate influenced both the rate and extent of b Dialyzed 64 hr against water at 4 C. c Protein was precipitated from the 20,000 X g germination. This was indicated by an experiment supernatant solution with (NH4)2SO4 (90%;satu- in which an equal number of heat-shocked spores ration) and centrifuged; the pellet was dissolved in (.4 X 104 spores per ml) were added to MYPG the original volume of water. 1082 COSTILOW AND COULTER APPL. MICROBIOL. TABLE 4. Effect of pyruvate oni the germinationi of DISCUSSION Rn7ri/lhiv nnnilli.,jo (??nQMA/ cnnrov nul1illus pupt1ilu _ZJV71V1)J JXu We have never been able to achieve the fre- l,xpt' quency of spore formation by B. popilliae strain 2309M in vitro as reported by Sharpe et al. (22). MIedium' I They obtained about 20% spores in this medium, (thou- II sands/ (millions/ml) (millions/ml) whereas we routinely observed 2 to 6°,0. However ml) they pointed out that the lot of yeast extract used influences the results dramatically. Also, they MYPGb 0.5 ...... 1.2 routinely prepared inocula by drying, whereas MYPG + pyr- (7.7%)c we either heat-shocked or exposed inocula to uvate (0. 1%) 67.5 9.4 (60.0%0, ) 3.6 (18%) chloroform to eliminate vegetative cells. This TYGb ...... 4.8 1.6 (10.3% ) could also TYG + pyru- influence the degree of sporulation. vate (0.1%).. 5.7 (29%) The generation time of B. popilliae cells grow- Brain heart in- ing in colonies on an agar medium is somewhat fusion (BHI). 2. 4 (12%) longer than that in broth. However, the greatest BHI + pyru- difference in development is in the period of time vate (0.1%).. 4.1 (21%) during which the growth rate is deaccelerating. This period is very prolonged when the cells are a Spores were heated at 60 C for 10HmC1 in ex- growing in colonies (3 to 4 days), as compared to periment I; they were treated with Cl[Cr20 min ex- a few hours in broth cultures (11). This difference periment II, and were heated at 60 C ft)r 20 mm in is probably due to the slow diffusion of the energy experiment III. I MYPG, 1% Mueller Hinton broth medium source to the cells on the agar. Glucose disap- solids (Difco), 1% yeast extract (Diifco), 0.3%7o peared from the agar at essentially the same time K2HPO4, and 0.05% glucose. TYG, Trypticase- that growth ceased. It is apparent that spore for- yeast extract-glucose. mation occurs with the 2309M strain during the c Percentages are of the number of slpores in the period of a declining growth rate, and we found original suspension determined by a diirect micro- no increase in the percentage of cells with spores scopic count. after this point. There was, however, a secondary growth response after some apparent cell lysis. and MYPG plus pyruvate broths ancI incubated These observations correlate well with those of on a shaker, and heat-shocked subsa,mples were Sharpe et al. (22). They observed that the spores plated at different intervals on the sarme medium in the colonies were found in a concentric ring containing pyruvate. The germinationi rates were near the outer edge of the colony surrounded by low in both media. Thus, the time reciuired for a vegetative cells and prespore forms in the outer 90%ZO reduction in the number of heat-resistant fringe. spores in the control broth was about X5 hr as com- It is known that some species of the pared to 4 hr in the broth plus pyruvi ate. After 3 sporulate in the presence of high levels of certain days of incubation of these media, aibout 1.1% energy sources (5, 11). Hsu and Ordal (5, 6) (350 per ml) of the original number of colony- demonstrated that Clostridium thermosaccharolyt- icum well when were forming units were still heat-resistant in the con- sporulated carbohydrates in thepyru-present which limited the growth rate or when trol as compared to 0.2% (75 per ml) iin the pyru- glucose was fed to the culture at growth-limiting vate medium. rates. Apparently, this organism requires carbo- The in vitro spores of 2309M are atctivated by hydrate for energy during sporulation. Sharpe et heat. A suspension of cells and spore2s was har- al. (22) observed that the presence of trehalose at vested from a plate ofMYPG after 23 dlays of incu- levels above 0.1 % greatly reduced the frequency bation and plated on JB medium (18) before and of sporulation with B. popilliae. However, high after heating at 60 C for 15 min. There were fewer populations of spores accumulate in the hemo- than 104 colony-forming units per ml in the un- lymph of the Japanese beetle larvae in the pres- of levels of trehalose It is heated samples compared to 8.3 x 105 in the ence high (17). possible, of course, that the trehalose heated. However, the spores are not verye heat- present is mostly Yhepat not available to the cells. Data in this paper indi- stable. After heating at 60 C for 20 min, the plate cate that spore formation occurs in a glucose me- count on MYPG plus pyruvate agar of a spore dium when the glucose levels become very low. suspension was 8.6 X 106 as compared to 3.1 X However, the percentage of cells forming spores in 101 and 1.4 X 106 after heating at 80 C'for 10 and the presence of a-methyl-D-mannoside was at least 20 min, respectively. equivalent to that in a glucose medium. A signifi- VOL 22, 1971 OLIGOSPOROGENOUS STRAIN OF B. POPILLIAE 1083 cant growth response was observed with increasing There is a great amount of evidence that the cata- levels of this sugar. The oxidation of a-methyl-D- lase activity in B. popilliae is sporulation specific; mannoside is inducible in this organism, and it is namely, (i) no activity is observed with 2309M utilized at much slower rates than glucose (Bhu- until m: rphological changes characteristic of miratana and Costilow, unpublished data). On the spore formation are observable, (ii) strains which basis of these facts, we propose that B. popilliae do not produce spores, including two variants requires the presence of a carbohydrate at concen- derived from 2309M, do not produce catalase trations which severely limit growth or in a when grown under the same conditions as 2309M, growth-limiting form for sporulation to occur. and (iii) all of the catalase activity in sporulating The pH changes in the medium during growth cultures of 2309M is stable to 80 C for 10 min. and sporulation of 2309M are quite similar to The catalase activity of B. popilliae was inhib- those observed during sporulation of other bacilli ited partially by heme poisons, cyanide and azide, (13, 26). However, the pH is not depressed as and, thus, appeared to be primarily due to a heme- much and rises to higher levels than observed with iron enzyme. However, its production was not other bacilli. In addition to oxidizing accumulated stimulated by the addition of preformed iron- acetic acid, basic compounds such as ammonia porphyrin compounds, hemin or boiled blood, as must either be produced or released from the cells. in the case of lactic acid bacteria (7). The activity Previous data demonstrated that this strain along was not reduced by exhaustive dialysis or by pre- with other oligosporogenous strains as well as cipitation with ammonium sulfate. some nonsporulating cultures would oxidize The germination of B. popilliae spores is quite acetate and glutamate (10). However, we have slow; 4 to 5 hours is required to reduce the num- never observed strain 2309S to oxidize these sub- ber of heat-resistant colony-forming units by strates, yet the changes in pH were similar in this 90% in a rich medium. Activation of the spores medium. by the procedure of Splittstoesser and Farkas (24) We were unable to demonstrate the appearance used for spores produced in vivo had no signifi- of extracellular protease or the production of an cant effect on the extent of germination. The addi- antibiotic by the oligosporogenous strain of B. tion of pyruvate to the germinating medium en- popilliae. These are early events commonly associ- hanced both the rate and extent of germination. ated with sporulation (13, 26). However, our data The presence of this compound in the plating me- are subject to question because ofthe relatively low dium enhanced recoveries by about 2- to over frequency of spore formation obtained. As 100-fold in different trials. However, the percent- pointed out by Schaeffer (21), oligosporogenous age of spores present which germinated varied cultures of bacilli may have phenotypes similar greatly among experiments and may have resulted to cultures which sporulate at high frequencies or from differences in the age of the suspensions. It to those which form no spores. However, a recent has been demonstrated that spores produced in report indicates that Bacillus brevis sporulates vivo germinate more readily after extended stor- normally without protease production or signifi- age (24). cant protein turnover (23). The only unique marker found which was asso- ACKNOWLEDGMENTS in vitro formation was the This research was conducted under a contract with the North- ciated with spore pro- ern Utilization Research and Development Division, Agricultural duction of a heat-stable catalase. Vegetative cells Research Service, U.S. Department of Agriculture, Peoria, Ill. of B. popilliae have no catalase activity (15, 25) We express our grateful appreciation to a number of scientists and they are sensitive to H202 (3), but spores from at the above named organization for their cooperative effort. bodies do have this Particularly, we thank Grant St. Julian, Jr., for supplying us larvae and refractile activity with spores of Bacillus popilliae from Japanese beetle larvae, (12). Catalase was consistently found at the onset E. S. Sharpe for providing us with strain NRRL B-2309M and of sporulation of 2309M. It appeared to be pro- with significant lots ofcells and spores produced in vitro, and R. A. duced at an early stage in spore formation since Rhodes for many stimulating discussions. In addition, the above in cells from This individuals along with W. C. Haynes and G. R. Hrubant saved it was present separated spores. us from much useless effort by ascertaining that a sporogenic is one of the first enzymes demonstrated in spores culture which we isolated and thought to be a variant of B. of aerobic bacilli (8). The catalase found in spores popilliae was actually a contaminant. We acknowledge the tech- of B. cereus was stable to 80 C for 10 min and 100 nical assistance of Kathleen Johnson and Loretta Laycock. C for 5 min, whereas vegetative cell enzyme was LITERATURE CITED inactivated these treatments. Sadoff completely by 1. Bray, G. A. 1960. A simple efficient liquid scintillator for (19) observed that the heat stability of catalase in counting aqueous solutions in a liquid scintillation counter. B. cereus developed at essentially the same time Anal. Biochem. 1:279-285. 2. Charney, J., and R. N. Tomarelli. 1947. A colorimetric as glucose dehydrogenase. This is one of the early method for the determination of the proteolytic activity sporulation-specific enzymes found in B. cereus. of duodenal juice. J. Biol. Chemii. 171:501-505. 1084 COSTILOW AND COULTER APPL. MICROBIOL.

3. Costilow, R. N., C. J. Sylvester, and R. E. Pepper. 1966. 15. Pepper, R. A., and R. N. Costilow. 1965. Electron transport Production and stabilization of cells of Bacillus popilliae in Bacillus popilliae. J. Bacteriol. 89:271-276. and Bacillus lentimorbus. Appl. Microbiol. 14:161-169. 16. Pheil, C. G., and Z. J. Ordal. 1967. Sporulation of the "ther- 4. Friedmann, T. E., and G. E. Haugen. 1943. Pyruvic acid. mophilic anaerobes. AppI. Microbiol. 15:893-898. II. The determination of keto acids in blood and urine. J. 17. Rhodes, R. A. 1967. Milky disease of the Japanese beetle, Biol. Chem. 147:415-442. p. 85-91. In Proc. Joint U. S.-Jap. Sem. Med. Control 5. Hsu, E. J., and Z. J. Ordal. 1969. Sporulation of Clostridium Insect Pests. Shukosha Printing Co., Ltd., Fukuoka, Japan. thermosaccharolyticum. AppI. Microbiol. 18:958-960. 18. Rhodes, R. A., E. S. Sharpe, H. H. Hall, and R. W. Jackson. 6. Hsu, E. J., and Z. J. Ordal. 1969. Sporulation of Clostridium 1966. Characteristics of vegetative growth of Bacillus thermosaccharolyticum under conditions of restricted popilliae. Appl. Microbiol. 14:189-195. growth. J. Bacteriol. 97:1511-1512. 19. Sadoff, H. L. 1961. Some properties of the spore catalase 7. Johnson, M. A., and E. A. Delwiche. 1965. Distribution and and some heat resistant enzymes in bacterial glucose de- characteristics of the catalases of Lactobacilliae. J. Bacteriol. hydrogenase. In H. 0. Halvorson (ed.), Spores IL. Burgess 90:347-351. Publishing Co., Minneapolis. 8. Lawrence, N. L., and H. 0. Halvorson. 1954. Studies on the 20. Sadoff, H. L., E. Celikkol, and H. E. Engelbrecht. 1970. spores of aerobic bacteria. IV. A heat resistant catalase Conversion of bacterial aldolase from vegetative to spore from spores of Bacillus terminalis. J. Bacteriol. 68:334-337. form by a sporulation specific protease. Proc. Nat. Acad. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Sci. U.S.A. 66:844-849. Randall. 1951. Protein measurement with the Folin phenol 21. Schaeffer, P. 1969. Sporulation and the production of anti- reagent. J. Biol. Chem. 193:265-275. biotics, exoenzymes, and exotoxins. Bacteriol. Rev. 33:48- 10. McKay, L. L., A. Bhumiratana, and R. N. Costilow. 1971. 71. Oxidation of acetate by various strains of Bacillus popil- 22. Sharpe, E. S., G. St. Julian, Jr., and C. Crowell. 1970. Char- liae. Appl. Microbiol. 22:1070-1075. acteristics of a new strain of Bacillus popilliae sporogenic 11. Majumder, S. K., and M. C. Padma. 1957. Screening of in vitro. Appl. Microbiol. 19:681-688. carbohydrates for sporulation of bacilli in fluid medium. 23. Slapikoff, S., J. L. Spitzer, and D. Vaccaro. 1971. Sporula- Can. J. Microbiol. 3:639-642. tion in Bacillus brevis: studies on protease and protein 12. Mitruka, B. M., R. N. Costilow, S. H. Black, and R. E. Pepper. tumover. J. Bacteriol. 106:739-744. 1967. Comparisons of cells, refractile bodies, and spores of 24. Splittstoesser, D. F., and D. F. Farkas. 1966. Effect of cations Bacillus popill ae. J. Bacteriol. 94:759-765. on activation of Bacillus popilliae spores. J. Bacteriol. 13. Murrell, W. G.i 1967. The biochemistry of the bacterial 92:995-1001. endospore, p. 1 33-251. In A. H. Rose and J. F. Wilkinson 25. Steinkraus, K. H. 1957. Studies on the milky disease organism. (ed.), Advances in microbial physiology, vol. 1. Academic II. Saprophytic growth of Bacillus popilliae. J. Bacteriol. Press Inc., New York. 74:625-632. 14. Neish, A. C. 1952. Analytical methods for bacterial fer- 26. Vinter, V. 1969. Physiology and biochemistry of sporulation, mentations. Report no. 46-8-3, 2nd ed. Prairie Regional p. 73-123. In G. W. Gould and A. Hurst (e&), The bac- Laboratory, Saskatoon, Canada. terial spore. Academic Press Inc., New York.