comparable results, were obtained with Bacillus cereus, Bacillus megatherium, ANTISPORULATION FACTORS IN COMPLEX ORGANIC MEDIA

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1 ANTISPORULATION FACTORS IN COMPLEX ORGANIC MEDIA II. SATURATED FATry ACIDS AS ANTISPORULATION FACTORS1' 2 W. A. HARDWICK, BEVERLY GUIRARD, AND J. W. FOSTER Department of Bacteriology, University of Texas, Austin, Texas Received for publication October 30, 1950 It has been demonstrated that antisporulation factors (ASF) for bacteria are present in the usual complex organic media and that such media may be rendered more sporogenic by treatment with adsorbents such as starch or activated charcoal (Roberts and Baldwin, 1942; Foster, Hardwick, and Guirard, 1950). This paper summarizes purification studies on ASF and identifies a part of the antisporulation activity of complex organic media as saturated fatty acids. The antisporulation activity of various pure known fatty acids is also reported. METHODS OF ASSAY An assay for the ASF was developed using Bacillus mycoides as the assay organism. A glutamic acid, salts, glucose medium was used, and cultures and spore-counting methods were essentially those described previously (Foster and Heiligman, 1949a). Graded amounts of the samples to be tested were added; and the smallest weight that would completely inhibit sporulation, while at the same time allowing maximutm growth, was chosen as the antisporulation activity of the sample. The departmental strain of B. mycoides was selected because of its large cell and spore size, making the spore counts easier. Also, this organism gives high spore yields (90 per cent) in 72 hours in control media with no ASF. All assays were incubated with continuous shaking. Frequent checks, with comparable results, were obtained with Bacillus cereus, Bacillus megatherium, and Bacillus globigii as assay organisms. EXPERIMENTS AND RESULTS Several attempts to elute the antisporulation activity that had been adsorbed from solutions of peptone (Foster, Hardwick, and Guirard, 1950) on activated charcoal (norit A) gave unsatisfactory results. Therefore other methods of extracting and recovering the ASF were investigated. Continuous ether extraction of various complex medium ingredients, such as peptone and "casamino acids" (in the dry state and in solution at different hydrogen ion concentrations), did not remove significant amounts of ASF. However, considerable amounts of antisporulation activity could be separated 1 This paper reports research undertaken in co-operation with the Quartermaster Food and Container Institute for the Armed Forces. The views or conclusions contained in this report are those of the authors. They are not to be construed as necessarily reflecting the views or having the indorsement of the Department of the Army. 2 Supported in part under a grant-in-aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. 145

2 146 W. A. HARDWICK, B. GUIRARD, AND J. W. FOSTER [VOL. 61 from the dry solids by continuous extraction with ethyl acetate, and to a lesser extent with ethyl alcohol or pyridine. Even though the activity was not etherextractable, the properties of the ethyl acetate extracts suggested the presence of lipidlike material. The insolubility in ether indicated that the ASF may be present in a bound form in the original complex organic medium, possibly as a lipoprotein, phospholipid, or cerebroside. Saponification with alcoholic KOH released substantially larger amounts of extractable antisporulation activity than was obtainable from the unsaponified material. After preliminary survey experiments a sample of commercial peptone, which was asporogenic even when fortified with a mineral salts mixture (Foster and Heiligman, 1949b), was selected for fractionation studies. The saponification and subsequent isolations of the fatty acid and unsaponifiable fractions were conducted according to the procedure of Hilditch (1947). TABLE 1 Antisporulation activity of lipid fractions obtained from a commercial peptone ORIGINAL PEPTONE PEPTONE RESDUE* VOLATILE FATTY NONVOLATILE FATTY UNSAPONIFIABLE ACID FRACTION ACID FRACTION FRACTION Lowest concentration completely inhibiting sporulation without significantly inhibiting vegetative growth 8g/ml pg/i pg/mi pg/mi pg/mi 15,000 60, Lowest concentration completely inhibiting vegetative growth 50, , Bacillus mycoides was the test organism. * Peptone fraction remaining after saponification and removal of lipids. The results of assays of these fractions from the peptone are reported in table 1. All three lipid fractions display much greater antisporulation activity than the original peptone and a substantial concentration of the physiologically active material from the peptone has been achieved in these lipid fractions. The removal of the lipid in each case leaves the residual peptone markedly detoxified with respect to sporulation. As shown in table 1 these fractions are inhibitory to vegetative growth at higher levels than those required to inhibit sporulation completely. The average yields of the lipid fractions per gram of original peptone from four separate experiments were as follows: unsaponifiable, 5.0 mg; nonvolatile fatty acids, 5.5 mg; and volatile fatty acids, 1.5 mg. The unsaponifiable fraction, presumably consisting mainly of sterols, had less antibacterial and less antisporulation activity than either of the fatty acid fractions. The antibacterial activity of certain sterols, e.g., cholesterol and ergosterol, is well known (Wynne and Foster, 1950). Possession of high antisporulation activity by both the volatile and nonvolatile fatty acid fractions indicated that this property is probably shared by several

3 1951] ANTISPORULATION FACTORS IN COMPLEX ORGANIC MEDIA 147 different fatty acids. Fractionation of the fatty acid extracts by means of paper chromatography was attempted, 1.5 mg solids being applied per initial spot. The developing solvent was water-saturated butanol. Duplicate strips were prepared, one being sprayed with bromeresol green to reveal the acidic areas as yellow spots on a blue-green background. The other paper strip was divided into five sections, corresponding to the acid spots revealed by the indicator, in the region of Rf values 0.1, 0.4, 0.7, 0.9, and 0.95, and each section was eluted by digesting it with alcohol. The ethanol eluates were evaporated to dryness; then each was made to 10 ml with water; and graded amounts of the eluates were assayed for antisporulation activity. The results of this preliminary work may be summarized as follows: the nonvolatile fractions had more antisporulation potency than the volatile fractions and the potency was roughly proportional to the Rf values. The following results were obtained with the volatile fatty acid fraction: at the 2.0-ml assay level the eluates from paper chromatograph sections with average Rf values of 0.1 and 0.4 caused no inhibition of sporulation of B. mycoides, that of 0.7 caused 15 per cent inhibition, that of 0.9 caused 63 per cent inhibition, and that of 0.95 caused 100 per cent inhibition. Vegetative growth was not significantly reduced in any of these treatments. A similar experiment using the nonvolatile fatty acid fraction gave the following sporulation inhibition values when assayed at the 1.0-ml level: eluates from paper chromatograph sections with average Rf values of 0.1, 0.4, 0.7, 0.9, and 0.95 gave sporulation inhibition of 0, 0, 0, 100, and 90 per cent respectively. The higher antisporulation activity of the nonvolatile acid fraction as compared to the volatile was confirmed by another experiment. This time some known saturated fatty acids were tested as controls. Particular emphasis was placed on the saturated acids because the conventional bromine test for unsaturation was completely negative on bothi the volatile and nonvolatile fatty acid fractions. Certainly the most potent of the known fatty acids tested with respect to antisporulation activity were those of intermediate carbon-chain length: capric (C10), lauric (C12), tridecylic (C13), and myristic (C14). The potency of acids with longer carbon chains drops off sharply. The relatively low potency of the volatile fatty acid fraction (consisting of Ci through C7 acids) agrees with the relatively low potency displayed by authentic relatively pure samples of volatile fatty acids. The high antisporulation potency of the nonvolatile fatty acid fraction (consisting of Cg through C18 acids) agrees both in Rf value and in antisporulation activity with the pure acids of intermediate carbon-chain length: capric, lauric, tridecylic, and myristic. In other experiments the C16 and C18 unsaturated fatty acids, oleic, linoleic, and ricinoleic acids, were tested. Oleic acid had much less antisporulation activity than the C10 to C14 acids. Linoleic and ricinoleic possessed definite antisporulation activity, the latter being more potent in this respect and approximately equal to the C1l to C14 acids. One striking difference between the action of linoleic and ricinoleic acids and that of the saturated acids is the fact that growth is always markedly inhibited by antisporulation concentrations of the former, whereas a

4 148 W. A. HABDWICK, B. GUIRARD, AND J. W. FOSTER TABLE 2 Antisporulation activity of known fatty acids [VOL. 61 NO. O CABON ATOIM AMOUNT INIBITING SPORULATION Butyric V... aleri e Caproic Heptylic Caprylic Pelargonic Capric Laurie Tridecylic Myristic Palmitic Stearic a oo25 ~.)> c c >4~~~~~~~~b bi 4 a~~~~~- 0 64< b.4'-.001~~~~~~~q >4 0 04~~ a C 0~~~~~.003cC Figure 1. Chart showing inhibition of sporulation and growth by fatty acids. The bars a-b represent the range over which the fatty acids inhibit sporulation: a represents the smallest concentration affecting sporulation; b represents the concentration above which growth as well as sporulation is inhibited; c is the concentration completely inhibiting growth.

5 1951] ANTISPORULATION FACTORS IN COMPLEX ORGANIC MEDIA selective influence on sporulation over growth is always characteristic of the saturated acids at the appropriate levels. Nevertheless, it is emphasized that all the fatty acids studied here do inhibit growth in relatively low concentrations, but definitely well above those concentrations exhibiting antisporulation activity. There was no evidence that the reduced antisporulation activity of palmitic, stearic, and oleic acids was due to lower solubility of these compounds. Data comparing the effect of different concentrations of various known fatty acids on growth and sporulation of the assay strain of Bacillus mycoides are given in figure 1. The particular peptone used in this work is, according to the manufacturer's literature, an enzymatic digest of casein. This makes it very likely that residual amounts of bound milk fat represent the source of the fatty acids found. Milk fat contains the following percentages of fatty acids: butyric, 3.0; caproic, 1.4; caprylic, 1.5; capric, 2.7; lauric, 3.7; myristic, 12.1; palmitic, 25.3; stearic, 9.2; arachidic, 1.3; and oleic, 29.6 (Hilditch and Longnecker, 1938). All of these acids have been demonstrated to possess some antisporulation activity, and the high potency of the nonvolatile fatty acid fraction very probably is due to the appreciable combined content of lauric and myristic acids (15.8 per cent) in milk fat. These were the two most potent fatty acids tested for antisporulation activity. Failure to detect the presence of oleic acid in the nonvolatile fraction by the bromine unsaturation test may be due to oxidation of this compound between the time the peptone was produced and the time it was analyzed, or during the extraction process. Antisporulation factors liberated by saponification were found in all of the commercial complex organic media or medium ingredients tested (table 3). It is of interest to note that the order of magnitude of activity is the same for the fatty acid fractions isolated from all the commercial products. Self-limitation of sporulation. If several cultures of sporulating bacilli are cultivated in a single medium, or if a single culture is inoculated into several different media, one observes large differences in degrees of sporulation among the various cultures. A number of different factors may be responsible for this observation (Foster, Hardwick, and Guirard, 1950). Now that fatty acids have been implicated in this respect it would appear that the ability of cultures to generate free fatty acids during their growth might lead to suppressive action on the subsequent sporulation of the culture. The degree to which this condition would develop and the differences between cultures theoretically depend on (1) the ability of the organism to synthesize fatty acids, and (2) the ability of the organism to liberate fatty acids that inhibit sporulation from the combined form in which they may be present in the medium. Obviously the strain of organism and the nature of the particular medium would be influential in this system. An experiment was performed demonstrating that a large increase in the amount of antisporulation lipid material occurs as a result of growth of two different sporulating bacilli in a peptone medium. Separate experiments showed that free lipid synthesis of B. mycoides in a glucose mineral salts medium was 149

6 150 W. A. HARDWICK, B. GUIRARD, AND J. W. FOSTER [VOL. 61 negligible insofar as antisporulation activity is concerned. Hence we may assume that the free lipid formation in the peptone medium represents, in part at least, release of initially present bound lipid, although the possibility of stimulation of lipid synthesis in the peptone medium is not excluded. The weight of the ether extracts at ph 3.0 was used as a gauge of free lipid in the peptone medium. Antisporulation TABLE 3 activity extractable by ether after saponification dehydrated organic media of several ETHER EXTRACT commercial ~ ~ ~ ~ ~ ~ -TOTAL INHIBITION XZ]DrUM Total Amount UNITSt wicdrecovered sinhibiting mg pg/m Of. assay medium Wilson liver fraction "B" ,400 Brain heart infusion (Difco) ,800 Neopeptone (Difco) ,600 Tryptone (Difco) ,500 Casamino acids (Difco) ,800 Peptone "C" (Albimi) ,000 Meat infusion (Difco) ,000 N-Z-amine (Sheffield) ,000 * From 40 g of commercial dehydrated medium. t An inhibition unit is defined as the smallest amount of antisporulation material per ml of assay medium that completely inhibits sporulation during the 72-hour incubation period. TABLE 4 Formation of antisporulation activity in a peptone medium as a result of autoclaving and growth of bacteria MEDrUM TREATMZNT ETHER EXTRACT INHIITION UNTSt mg Before autoclaving After autoclaving Centrifugate from 72-hr B. cereus culture 320 3,200 Centrifugate from 72-hr B. mycoides culture 400 4,000 * From medium equivalent to 25 g peptone as 1.5 per cent solution. t See second footnote of table 3. Table 4 shows that autoclaving the medium increases by 5-fold the amount of free lipid, this increase doubtless representing release from bound forms. However, a further marked increase in free lipid content of the mediuim occurred as a result of separate growth of B. cereus and of B. mycoides. The data given in table 4 apply to the bacteria-free centrifugates remaining after centrifugation of the culture. The antisporulation activity of the ether extracts was assayed in each case. A marked increase in the total number of antisporulation units

7 1951] ANTISPORULATION FACTORS IN COMPLEX ORGANIC MEDIA 151 was obtained in the medium as a result of the growth of the bacteria. This generation of antisporulation activity may affect in a significant way the sporulation behavior of some bacilli under certain conditions. SUMMARY The volatile and nonvolatile fatty acid fractions obtained from a commercial peptone inhibit sporulation of aerobic bacilli in a synthetic medium at concentrations not inhibitory to growth. The nonvolatile fatty acid fraction is more active in this respect. The major portion of lipid material in complex organic media exists in bound form and can be released by saponification. Data for several media are given. Unsaturated fatty acids could not be detected in the volatile and nonvolatile acid fractions. Several pure known saturated fatty acids possess antisporulation activity; the acids of intermediate carbon-chain length, namely, capric, lauric, tridecylic, and myristic acids, are outstanding in this respect. The high activity of the nonvolatile fatty acid fraction isolated from the peptone probably is due to the lauric and myristic acids it contains, as these together account for 15.8 per cent of milk fat. The particular peptone fractionated is a casein digest, and doubtless contains residual bound milk fat. In complex organic media the bound lipid and the component fatty acids are released during the growth of bacteria so that cell-free growth medium from cultures contains appreciable quantities of antisporulation fatty acids. This might be responsible for some degree of self-limitation of sporulation of bacteria. Autoclaving uninoculated media increases the amount of ether-soluble material that is inhibitory to sporulation. REFERENCES FOSTER, J. W., HARDWICK, W. A., AND GUIRARD, BEVERLY 1950 Antisporulation factors in complex organic media. I. Growth and sporulation studies on Bacillus larvae. J. Bact., 59, FOSTER, J. W., AND HEILIGMAN, F. 1949a Biochemical factors influencing sporulation in a strain of Bacillus cereus. J. Bact., 57, FOSTER, J. W., AND HEILIGMAN, F. 1949b Mineral deficiencies in complex organic media as limiting factors in sporulation of aerobic bacilli. J. Bact., 57, HILDITCH, T. P The chemical constitution of fats. 2d ed. John Wiley & Sons, New York. Refer to p HILDITCH, T. P., AND LONGNECKER, H. E Further determination and characterization of the component acids of butter fat. J. Biol. Chem., 122, ROBERTS, J. L., AND BALDWIN, I. L Spore formation by Bacillus 8ubtilis in peptone solution altered by treatment with activated charcoal. J. Bact., 44, WYNNE, E. S., AND FOSTER, J. W Studies on the effects of C18 unsaturated fatty acids on growth and respiration of Micrococcus pyogenes var. aureus. J. Infectious Diseases, 88,