NUTRITIONAL REQUIREMENTS FOR VEGETATIVE GROWTH OF MYXOCOCCUS XANTHUS MARTIN DWORKIN' Department of Microbiology, Indiana University Medical Center, Indianapolis, Indiana Received for publication February 8, 1962 ABSTRACT lation and germination in Bacillus have been shown to be nutritionally dependent (Hardwick DWORKIN, MARTIN (Indiana University Medi- and Foster, 1952; Hills, 1950). The formation of cal Center, Indianapolis, Ind.). Nutritional re- perithecia in Melanospora can be regulated by the quirements for vegetative growth of Myxococcus nature and concentration of various constituents xanthus. J. Bacteriol. 84:250-257. 1962.-This in- of the medium (Asthana and Hawker, 1936), and vestigation was part of a program to clarify the fruiting-body formation by Myxococcus (Oetker, environmental regulation of fruiting-body forma- 1953) and Chondromyces (Kiuhlwein, 1950) has tion in a fruiting myxobacterium. By use of a been shown to depend upon the nature of the dispersed-growing strain of M1yxococcus xanthus, medium. The clarification of this influence is the nutritional requirements for vegetative likely to lead to an understanding of the nature of growth have been defined. Exponential growth the interaction between the environment and the will take place on a medium containing 17 amino regulatory mechanisms controlling morpho- acids and salts. The generation time is 8 to 10 hr. genesis. Various optima have been established. There are Although a number of investigators have ex- no requirements for exogenous vitamins, and, amined the stimulatory or inhibitory effect of with the exception of glycogen, a variety of various nutrilites (Nor6n, 1955; Oxford, 1947) organic materials do not significantly stimulate and the suitability of various complex media growth. (Oetker, 1953), there has been no clear definition of the nutritional requirements of any of the fruit- It is unrealistic to hope that one can critically ing myxobacteria. To a large extent, investiga- study the relationship between an organism and tions in this area have been restricted by the lack its environment without controlling the nu- of a good quantitative method for measuring tritional milieu. In general, physiological studies myxobacterial growth. Growth occurred either as of microorganisms should be carried out, when- a ring around the culture tube, in which case the ever possible, in chemically defined media. thickness of the ring was measured (Nor6n, 1955), Specifically, when investigating the effect of the or as colonies on solid media, in which case colony environment on microbial morphogenesis, this diameter was the parameter of growth (Oetker, must be a sine qua non. Moreover, an understand- 1953). The use of dispersed-growing variants of ing of the nutritional requirements of an organ- Myxococcus by Loebeck and Klein (1956) and by ism is apt to provide insight into a number of Holt (1960) paved the way for an analytical ap- facets of the nature of that organism. To a large proach to the problem. extent, the ecological role of a microbe will be By use of such dispersed-growing variants, this related to its nutritional capacity, and an exami- investigation has been concerned with defining nation of its metabolic apparatus may be broadly the nutritional requirements for the vegetative guided by a knowledge of its nutritional needs. growth of Myxococcus xanthus. However, the most compelling reason for our MATERIALS AND METHODS interest in the nutrition of a fruiting myxobac- Organisms. Unless otherwise indicated, the data terium is the oft-described relationship between to be described were obtained with a variant of the nutritional milieu of a microbe and its mor- M. xanthus referred to as VC. This organism was phogenetic development. The processes of sporu- isolated by John Holt while at Purdue University. Present address: Department of Microbiology, Under appropriate conditions, VC grows in a dis- University of Minnesota, Minneapolis, Minnesota persed state and, as such, is suited for turbidi- 250 VOL. 84, 1962 NUTRITION OF MYXOCOCCUS XANTHUS 251 metric measurement of growth. After about 100 RESULTS serial transfers in complex liquid medium, this Growth on culture lost the ability to form either microcysts complex media. 1M. xanthus will grow or well on a medium containing enzymatic digests of fruiting bodies, while retaining its character- Its istic, gliding motility. Holt (1960) also observed protein. growth on CT medium is illustrated this in Fig. 1. The generation time is about 3.5 hr. phenomenon. The defined medium, as Under these the finally developed, was also tested for its ability to conditions, stationary phase is followed by rapid autolysis of the cells. The support the growth of two other strains of M. maximal Klett xanthus, designated FB and MC. Strain FB, ob- reading usually obtained is about a cell concentration tained from the stock culture collection of the 500, representing of about 2 X 109 organisms/ml. We have not been able to bacteriology department of the University of increase California in Berkeley, will significantly either the rate of growth or undergo the complete the maximal yield of cells, and have therefore developmental cycle, forming microcysts and the fruiting bodies. Strain adopted growth pattern on CT as the stand- MC was derived from FB, ard of measurement. The and will form microcysts but not mature fruiting principal parameter used for evaluating growth was the generation bodies. Both of these strains will grow in a dis- time during exponential multiplication. persed state. A number of Growth conditions. Dispersed growth of vegeta- protein hydrolyzates were com- tive cells was pared to Casitone. Although there was no striking obtained by inoculating 40 ml of CT difference the various medium in a 250-ml Erlenmeyer flask and in- among enzymatic digests of protein, the acid hydrolyzates were significantly cubating at 30 C (+1 C) with shaking (about 75 inferior to 100 (Table 1). Since acid hydrolysis of protein rotations per minute). CT medium con- will and the tains Casitone (Difco), 2%; destroy , effect of MgSO4, 0.1%; adding these amino acids (0.1 mg/ml) back to the K2HP04-KHY04, 0.01 M (pH 7.2); and distilled acid water. hydrolyzates (Casamino Acids and Acid- (Casitone is an enzymatic hydrolyzate of was tested. This not casein.) To avoid icase) did improve the suit- precipitation, the MgSO4 was ability of the acid hydrolyzates. The media added aseptically after the other components were containing the acid hydrolyzate contained a sig- autoclaved. Growth on CT agar is vegetative, nificant amount of sodium with no microcysts or chloride (0.38%). To fruiting bodies formed. For eliminate the possibility that microcyst or fruiting-body formation, a medium growth was in- prepared by autoclaving a suspension of Esch- erichia coli in distilled water (about 5 X 108 cells/ml) and agar (2%) for 1.5 hr was used. This medium is referred to as CA. Cultures were main- tained by daily transfer in CT liquid. With each transfer, the culture was streaked on a CT plate 0 and, in the case of MC and FB, also on a CA plate, z to maintain the ability to form microcysts and fruiting bodies. 0 For growth measurements, cultures were grown in 250-ml Erlenmeyer flasks with Klett tubes attached. Growth was measured with a Klett-Summerson colorimeter using the green (no. 57) filter. For nutritional experiments, the inocu- lum was prepared by centrifuging cells out of an 18- to 24-hr-old culture. The cells were resus- pended in distilled water, and the experimental flasks were inoculated to about 108 cells/ml.

Chemicals. All amino acids and were 0 10 20 30 40 50 60 70 80 90 100 obtained from the Cyclo Chemical Corp., Los T I M E IN HO UR S Angeles, Calif., as the L isomers unless otherwise FIG. 1. Growth of Myxococcus xanthus VC on stated. various media. 252 DWORKIN J. BACTERIOL. hibited by the presence of sodium chloride, an TABLE 2. Effect of addition of single amino acids to equivalent amount of sodium chloride was added a 0.5% Casamino Acids-salts medium to the CT medium. There was no inhibition of (0.5 mg/ml) Amino acid (0.5 mg/ml) growth. As an additional control, a 10% solution of Casamino Acids was electrically desalted and Stimulatory then tested. There was no change in its ability to f3- support growth. Asparagine Growth-factor requirements. Although Nor6n Carnosine (1955) indicated that growth of M. virescens was of a of vita- stimulated by the presence variety Homocystine mins, Oxford (1947) found that a vitamin mixture Inhibitory Indifferent had no effect on the growth of the same organism. Glutamine We were unable to demonstrate any stimulatory Tryptophan Djenkolic acid effect of vitamins on the growth of M. xanthus. In an attempt to detect a requirement for Alanine -y-Aminobutyric acid steroids or fat-soluble vitamins, 2 g of finely Cysteine *HCI powdered Casitone were placed in a Soxhlet ap- paratus and extracted for 48 hr with diethyl a-Aminobutyric acid ether. There was no alteration in the ability of the a-Aminoisobutyric Betaine Casitone to support growth. This would seem to acid j-Aminobutyric acid indicate the absence of any fat-soluble vitamin or DL- steroid requirements. The ability of vitamin-free Aspartic acid Cysteic acid Casitone (Difco) to support growth was tested. Glycocyamine This material is prepared from casein which is sufficiently free of vitamins to serve in assay media for nicotinic acid, thiamine, pantothenic 0.4,g/ml; nicotinamide, 0.4 Ag/ml; biotin, 0.01 acid, biotin, vitamin B12, and folic acid (Difco ,ug/ml; folic acid, 0.05 Ag/ml; B12, 0.005 19g/ml) Laboratories, 1953). Growth was equal to that had no stimulatory affect on growth. obtained with regular Casitone. After the defined Amino acid requirements. The superiority of the medium had been developed, the addition of a enzymatic hydrolyzate over the acid hydrolyzate vitamin mixture (riboflavine, 0.5 ,ug/ml; p-amino- implied that peptides were a better source of benzoic acid, 1.0 ug/ml; pyridoxal-PO4, 2,g/ml; amino acids than were the free amino acids them- thiamine-HCl, 0.1 Ag/ml; calcium pantothenate, selves. In view of the extreme difficulty in either duplicating the complex mixture of peptides in TABLE 1. Growth on various protein hydrolyzates Casitone or in isolating, identifying, and supply- Generation Highest Kiett ing the required peptides, an alternate approach Hydrolyzate (I1%) time reading was sought. This approach was suggested by the hr work of Kihara, Ikawa, and Snell (1961), who showed that the stimulation by peptides of the Enzymatic hydrolyzates growth of Streptococcus faecalis reflects not a re- Casitone (Difco) ...... 4 337 Trypticase (Difco)...... 5 419 quirement for the per se but an overcom- Tryptone (Difco) ...... 6 467 ing of an amino acid imbalance or antagonism. Milk protein hydrolyzate This imbalance presumably inhibits the entry of (BBL) ...... 6 381 the required amino acid into the cell. This view Phytone (BBL) ...... 7 361 has proven fruitful in clarifying the role of Lactalysate (BBL)...... 9 398 peptides in bacterial nutrition. Accordingly, our Peptone (Difco) ...... 10 234 approach was to attempt to duplicate the Acid hydrolyzates Casamino Acids mixture with known amino acids Casamino Acids (Difco and then to vary the levels of the component Certified) ...... 28 100* amino acids. However, all of our initial attempts Acidicase (BBL) ...... 44 75* to obtain any vegetative growth with mixtures of * Growth was still taking place. amino acids failed. VOL. 84, 1962 NUTRITION OF MYXOCOCCUS XANTHUS 253

TABLE 3. Effect of individual amino acids as acids were essential or stimulatory and which determined by elimination from the were indifferent. Table 3 lists those amino acids defined medium whose omission from the mixture reduced growth. Amino acid Optimal concn* Eight amino acids were required and nine were stimulatory. When all nine stimulatory amino mg/ml acids were eliminated, however, no growth took Required place. Leucine ...... 1.00 Without consideration of the possible concen- Isoleucine ...... 0.....50 Proline ...... 0 .50 tration interactions, concentration optima were Methionine ...... 0...... 05-0.50 determined by varing the concentration of single Alanine ...... 0.05-0.50 amino acids. The optimal concentration for a Valine ...... 0.10-0.50 number of amino acids was quite narrow. Glycine, Glycine ...... 0 .05-0.25 for example, was absolutely required for growth. Djenkolic acidt ...... 0.10-0.25 The optimal concentration was 50 ,ug/ml, and Stimulatory concentrations greater than 250 ,ug/ml completely Tyrosine ...... 0.60 inhibited growth. Table 3 lists for some amino Asparagine ...... O.50 acids the range of concentrations within which Arginine ...... 0.10 optimal growth occurred. For the other amino Histidine ...... 0.05-0.25 concentration Lysine ...... 0.25-0.75 acids, the optimal either varied from experiment to experiment or could not be Phenylalanine ...... 0.015-0.15 Threonine ...... 0.10 clearly defined. Serine ...... 0.10-0.50 On the basis of these experiments, we were able Tryptophan ...... 0.05-0.50 to construct a synthetic medium consisting of salts and 17 amino acids. The composition of this * Those amino acids for which a single concen- medium is described in Table 4. Growth on this tration is given demonstrated no well-defined medium is illustrated in Fig. 1. optimal range. Occasionally, a batch of medium would not sup- t In the absence of cystine.

Noren (1955) was able to demonstrate effects TABLE 4. Composition of defined medium of single amino acids on the growth of M. virescens Component Concn when the amino acids were added to a basal medium containing low levels of Casamino Acids. mg/ml We utilized this approach by adding single amino Tyrosine .. 6 acids at a level of 0.5 mg/ml to a limiting amount Asparagine .. 5 of Casamino Acids (0.5%). Under these con- Leucine . 1.0 ditions, distinct patterns of stimulation and in- Isoleucine . .5 hibition of growth were noted and it was possible Proline . .5 to determine the approximate level above which Arginine .0.1 Histidine . .05 any particular amino acid was toxic. Table 2 Glycine .. .05 tested and the effect of these lists the amino acids Lysine . .25 amino acids on the growth of vegetative cells in Methionine . .05 the presence of 0.5% Casamino Acids. Phenylalanine*.0.15 On the basis of these results, a synthetic Tryptophan* .0O.05 medium was then devised containing the stimula- Serine.0.1 tory and indifferent amino acids at levels of 0.5 Threonine .0.1 mg/ml and the inhibitory amino acids at levels of Valine.0.1 50 ,ug/ml (at which concentration they did not Djenkolic acid.0.1 Alanine* . .05 inhibit growth on 0.5% Casamino Acids). This Glycogen.3.0 mixture supported growth of VC. The rate of MgSO4.1.0 growth, however, was extremely low. Eliminating K2HPO4-KH2PO4 (pH 7.6) .0.01 M individual amino acids from this synthetic medium permitted us to determine which amino * Used at 1.0 mg/ml for FB and MC. 254 DWORKINOJ. BACTE:RIOI,.

TABLE 5. Effect on growth of the addition of various however, stimulate the production of a bright-red organic materials to the amino acids-salts medium extracellular pigment. This phenomenon was en- Addition (0.1%)* Addition (0.1%) countered on numerous occasions and could not be unequivocally related to the rate or amount of Marked stimulation Inhibition growth. Adding an ashed residue of Casitone to Glycogen (shellfish) Mannose the synthetic medium had no stimulatory effect Glycerol Galacturonic acid on growth. Slight stimulation Sodium citrate Optimal pH. There was no sharp pH optimum Fructose for vegetative growth. In the defined medium, Glucose Lauryl alcohol Arabinose Capric acid growth was inhibited at pH 6.8 and below, and Soluble starch Adenine sulfatet was maximal between pH 7.2 and 8.2. It must be Glucuronic acid Guanine kept in mind that the inhibitory effect of phos- Glucosaminic acid Indifferent phate precluded its use at concentrations higher Ammonium malate Maltose than 0.01 M. Its buffering capacity was therefore Succinic acid Sorbose quickly exceeded and the pH experiment meas- Uracil Cellobiose ured only the effect of the initial pH. Xanthine Sucrose Effect of organic materials. The effect on growth Yeast nucleic acid Trehalose of a wide variety of organic compounds was Raffinose were Xylose tested (Table 5). These materials added to Sodium acetate the amino acid-salts mixture. Although many of L-Tartaric acid the compounds tested showed a slight stimulatory effect (e.g., starch, uracil, succinic acid), glycogen * All carbohydrates, sugar acids, and glycerol was the most effective, reducing the generation were autoclaved separately and added aseptically. time from 11 to 8 hr. This is consistent with the t Added at 0.01%. observation of Nor6n (1955), who reported that port any growth. The only known appropriate M. virescens can utilize glycogen and starch. It variable was the occasional use of DL amino acids seemed at first unlikely that this was a nutritional rather than the L isomers. The inhibitory effect of effect due to the glycogen, since commercially the D isomer was investigated. Each amino acid available glycogen was found to be frequently required in the medium was supplied singly as contaminated with nitrogenous materials and the DL mixture at twice the usual concentration of since the optimal effect was at a rather high con- the L isomer, and the effect on growth was deter- centration (0.3%). Also, neither glucose nor mined. DL-Leucine completely inhibited growth, maltose showed any marked stimulatory effect. while DL-isoleucine caused a partial inhibition. However, glycogen was precipitated three times The effect of adding twice the normal level of from hot water by 95% ethyl alcohol and lost L-leucine or L-isoleucine was tested. In neither none of its growth-stimulating activity, despite a case did this result in equivalent inhibition. considerable reduction in ninhydrin-positive ma- Optimal concentration of inorganic components. terial. The optimal concentration of MgSO4 in the In another experiment, the fate of glycogen in synthetic medium was determined and was found a growing culture was followed by a quantitative to lie between 0.1 and 0.2%. No growth took anthrone test (Carroll, Longley, and Roe, 1956) place in the absence of MgSO4, and the growth for carbohydrates and a quantitative iodine test rate was decreased at 0.3 and 0.03%. The optimal (Van der Vies, 1954) for polysaccharide (Fig. 2). level of phosphate was 0.01 M. Growth was in- Determinations were made on the cells and on the hibited at 0.03 and 0.003 M. culture filtrate. The level of iodine- and anthrone- Two mixtures of trace elements [(i) Metals reactive material in or on the cells remained low "44" (Cohen-Bazire, Sistrom, and Stanier, 1957); and constant. In the filtrate, however, during the and (ii) MgSO4, 500 ,g/ml; MnSO4, 5 Ag/ml; early phase of growth there was a sharp decrease ZnSO4, 5 ,ug/ml; FeSO4, 10 ,g/ml; CaCO3, 2 in iodine-reactive material (polysaccharide). gg/ml] were tested with the synthetic medium There was no corresponding decrease in anthrone- and were found to exert no marked stimulatory reacting material, indicating the presence of non- effect on growth. The trace-element mixtures did, polymerized carbohydrates in the supernatant VOL. 84, 1962 NUTRITION OF MYXOCOCCUS XANTHUS 255 culture fluid. After about 1 day of growth, the various media is illustrated in Fig. 1. The gener- level of iodine-reacting material started rising, ation time obtained on CT was 3.5 hr; on terminating in a level of polysaccharide consider- Casamino Acids, 28 hr; on the amino acid ably higher than that found in a culture without medium, 11 hr; and on the amino acids-glycogen glycogen. medium, 8 hr. Effect of dipeptides. At this point it was neces- Growth of MC and FB. The amino acids-salts sary to consider the basis for the more rapid medium was tested for its ability to support growth on the Casitone medium (generation time vegetative growth of the MC and FB strains. of 3.5 hr) as compared to the synthetic medium Neither was able to grow under these conditions. (generation time of 8 hr). Since vitamins and trace When the levels of tryptophan, phenylalanine, elements had been effectively ruled out, the en- and alanine were raised to 1 mg/ml, growth took hanced growth was probably due to the superi- place with a generation time of 12.5 hr for both ority of the peptide as a source of amino acids. MC and FB. This was reduced to 10 hr in the Accordingly, single dipeptides were added (0.25 presence of 0.3% glycogen. The terminal growth mg/ml) to the complete amino acid medium; 36 obtained was considerably higher than that ob- dipeptides, containing all the required and stimu- tained with the VC variant. latory amino acids except djenkolic acid, were Growth on solid medium. Ultimately, it is our tested. A variety of peptides stimulated growth hope to study fruiting-body formation under con- but in no case was the rate of growth equal to ditions of defined nutrition. It was necessary, that obtained on CT. When all of the stimulatory therefore, to be sure that vegetative growth of peptides were added to the synthetic medium, FB would take place on solid media. When the growth was enhanced (generation time of 6.5 hr) defined medium was solidified with 1% agar but was still considerably inferior to that obtained (Oxoid) and inoculated with FB or VC, typical on CT. The stimulatory dipeptides were: methi- vegetative, colonial growth took place. No onine amide *HCl, alanyl-isoleucine, prolyl-gly- microcysts or fruiting bodies were formed. As in cine, prolyl-leucine, valyl-valine, a-glutamyl- the liquid medium, growth was enhanced by the tyrosine, tyrosyl-glycine, prolyl-tyrosine, phenyl- presence of 0.3% glycogen. alanyl-leucine, alanyl-phenylalanine, isoleucyl- Serial transfer in defined medium. It is important alanine, and isoleucyl-leucine. to demonstrate that growth in the defined After combining all of the above optima, vege- medium is not dependent on either the carry-over tative growth was obtained which, while inferior of complex material with the inoculum or on the to growth on CT, was superior to that on intracellular accumulation of some essential Casamino Acids. A comparison of growth on the nutrient present only in the complex medium. Ac- cordingly, serial passage in the defined medium (Table 4) was attempted and was successful. There was no change in the growth patterns of VC or FB after five transfers on the defined medium, with or without glycogen. a z

LM DISCUSSION :4 The principal purpose of this investigation has been accomplished, namely, the formulation of a of M. ,4 defined medium for vegetative growth -3 xanthus. Many questions, however, remain un- x answered. Unlike a simple glucose-salts medium, it is impossible at this point to decide what is serving as the energy source, source, and carbon source. Are some amino acids being oxi- or 0 10 20 30 40 50 60 dized while others are incorporated, do many T I M E I N H O U R S serve a dual function? The clarification of the FIG. 2. Relation between growth and glycogen util- roles of the individual components of the medium ization in Myxococcus xanthus VC. will have to await a more detailed metabolic 256 DWORKIN J. BACTERIOL. study. The fact that some amino acids are stimu- maltose, or by sugars in general, it is unlikely that latory rather than absolutely required may re- the function of the glycogen is to provide an ex- flect either a rate-limiting synthesis of the par- ternal source of sugar. The AF of phosphorolysis ticular amino acid or the presence of the amino of glycogen is +1.5 kcal, so it is unlikely that the acid as a component of the slime of the inoculum. process furnishes energy to the cell. In the pres- The latter factor may be responsible for the diffi- ence of glycogen, the final level of iodine-reactive culty encountered in establishing optimal concen- material in the culture filtrate is considerably trations for some of the amino acids in the higher than that found in the absence of glycogen. medium. Since this is preceded by a sharp decrease in The nature of the medium poses some questions. iodine-reactive material, it is likely that this final Magnesium sulfate is required at an unusually level represents polysaccharides synthesized by high concentration (0.1%), yet this was clearly the organism. Holt (1960) has shown that the shown to be the optimal level. Leucine, and in slime of M. xanthus contains about 20% poly- the case of substrains FB and MC, tryptophan, saccharide. The role of the slime in vegetative phenylalanine, and alanine are required at levels growth as well as in the formation of microcysts of 1 mg/ml. At lower levels, either growth does and fruiting bodies will undoubtedly prove to be not take place at all or the rate is considerably an important one. lower. These relatively high levels may be re- The ability to cultivate a fruiting myxobac- quired to overcome permeability difficulties or to terium under defined nutritional conditions places overcome the antagonistic effects among some the future goals of this investigation within reach. amino acids. This is consistent with the observa- Preliminary experiments indicate that proper tion that peptides serve the organism much more manipulation of the constituents of the defined effectively than the corresponding amino acids. It solid medium can control fruiting-body formation. is interesting that, despite the superiority of CT We are now in a position to examine the specific over the amino acid medium, adding dipeptides to relation between fruiting and the nutritional the amino acids does not significantly enhance milieu in the hope of clarifying the regulatory growth. Either the dipeptide is not sufficiently mechanisms which control fruiting-body forma- large or the relative position of the amino acid in tion. the dipeptide is critical. The superiority of djenkolic acid as a sulfur ACKNOWLEDGMENTS source is unusual. It was found to be superior to It is a pleasure to acknowledge the excellent cysteine, cystine, homocystine, and lanthionine. technical assistance of Charles E. Smith and Although djenkolic acid is not a component of John T. Russell. bacterial protein, it has been shown to serve satis- This investigation was supported by grants factorily as a sulfur source for Streptococcus bovis from Eli Lilly and Co. and the National Science (Prescott, 1961). The cell surface of M. xanthus Foundation (NSF G-10902). is sensitive to cysteine and to sulhydryl com- pounds in general, forming spheroplasts in their LITERATURE CITED and The relative presence (Dworkin Voelz, 1962). ASTHANA, R. P., AND L. E. HAWKER. 1936. The unsuitability of cystine may be a reflection of its influence of certain fungi on the sporulation of low solubility. The optimal concentration of Melanospora destruens Shear and of some other djenkolic acid is about twice the highest concen- Ascomycetes. Ann. Botany (London) 50:325- tration of cystine obtainable under the conditions 344. of culture. CARROLL, N. V., R. W. LONGLEY, AND J. H. ROE. The role of glycogen is unclear. Its function is 1956. The determination of glycogen in liver not solely a physical one, since it disappears from and muscle by use of anthone reagent. J. the medium. The appearance in the medium of Biol. Chem. 220:583-593. anthone-positive material which does not react COHEN-BAZIRE, G., W. R. SISTROM, AND R. Y. STANIER. 1957. Kinetic studies of pigment with iodine implies that the glycogen is hy- synthesis by non-sulfur purple bacteria. J. drolyzed extracellularly. Since the growth of the Cellular Comp. Physiol. 49:25-68. cells is not appreciably stimulated by glucose, DIFco LABORATORIES. 1953. Difco manual of de- VOL. 84, 1962 NUTRITION OF MYX4OCOCCUS XANTHUS 257

hydrated culture media and reagents for Entwicklungsgeschichte der Myxobacterien. microbiological and clinical laboratory pro- Arch. Mikrobiol. 14:678-704. cedures, 9th ed. Difco Laboratories, Detroit. LOEBECK, M. E., AND H. P. KLEIN. 1956. Sub- DWORKIN, M., AND H. VOELZ. 1962. The formation strates for Myxococcus virescens with special and germination of microcysts of Myxococcus reference to eubacterial fractions. J. Gen. xanthus. J. Gen. Microbiol. 28:81-85. Microbiol. 14:281-289. HARDWICK, W. A., AND J. W. FOSTER. 1952. On the NORtN, B. 1955. Studies on myxobacteria. III. Or- nature of sporogenesis in some aerobic bac- ganic factors in nutrition. Satryck ur Bo- teria. J. Gen. Physiol. 35:907-927. taniska Notiser. 108:81-134. HILLS, G. M. 1950. Chemical factors in the germi- OETKER, H. 1953. Untersuchungen uber die Er- nation of spore-bearing aerobes: observations nahrung einiger Myxobakterien. Arch. Mikro- on the influence of species, strain, and con- biol. 19:206-246. ditions of growth. J. Gen. Microbiol. 4:38-47. OXFORD, A. E. 1947. Observations concerning the HOLT, J. G. 1960. The nature of the slime of growth and metabolic activities of myxococci Myxococcus xanthus. Thesis, Purdue Uni- in a simple protein-free liquid medium. J. versity. Bacteriol. 53:129-138. KIHARA, H., M. IKAWA, AND E. E. SNELL. 1961. PRESCOTT, J. M. 1961. Utilization of sulfur com- Peptides and bacterial growth. X. Relation of pounds by Streptococcus bovis. J. Bacteriol. uptake and hydrolysis to utilization of D-ala- 82 :724-728. nine peptides for growth of Streptococcus VAN DER VIES, J. 1954. Two methods for the de- faecalis. J. Biol. Chem. 236:172-176. termination of glycogen in liver. Biochem. J. KtHLWEIN, H. 1950. Beitrage zur Biologie und 57:410-416.