Role of Amino Acids in Germination of Bacillus licheniformis Spores. II. Effect on Radioactive Spores1

ROLE OF AMINO ACIDS IN GERMINATION OF BACILLUS L I C H E N I F O R M I S SPORES. II. EFFECT ON RADIOACTIVE SPORES 1 J. H. MARTIN AND W. J. HARPER Department of Dairy Technology, The Ohio State University, Columbus ABSTRACT

The distribution of radioactivity in the cytoplasm, spore coats, and exudates during germination of Bacillus ticheniformis spores labeled with radioactive L-alanine and L-valine was determined. When the radioactive spores were germinated with nonradioactive L-alanine and L-valine, radioactivity was released after germination medium, with very little radioactivity released after germination was complete. Most of the radioactivity had its origin in the cytoplasm in L-alanine-induced germination, but with L-valine approximately 50% of the radioactivity was released from the spore coats. The radioactivity released was associated with ninhydrin-positive components on p a p e r chromatograms, one at the point of application to the paper, the other at the R f value of alanine. When n-alanine was used to germinate the spores, the greater portion of radioactivity (66%) was in the a]anine spot. When L-valine was the germinant, approximately 50% of the radioactivity in the exudates remained as a nonmobile ninhydrin-positive component. When radioactive spores were germinated with L-alanine, free D-alanine was released into the germination medium, with the concentration increasing throughout a 4-hr incubation period. D-alanine was not released when L-valine was used as the spore germinant. Spores germinated with either L-alanine or L-valine also released dipicolinie acid (DPA) into the germination medium after germination. The release of D P A was somewhat slower than that of D-alanine, with l)-alanine appearing in the germination exudates during the process of germination, and D P A being released only after germination was practically complete. Spores of Bacillus licheniformis have been shown previously to germinate readily in response to L-alanine, L-valine, and L-cysteine, whereas the D-forms of these amino acids inhibited germination (7). Uptake of the amino acids capable of inducing germination, as well as some which caused no germination, was equally rapid, indicating that the mechanism of amino acid-induced spore germination is not dependent on permeability differences in the dormant spore (8). Uptake and distribution studies with radioactive L-alanine and Lvaline suggested that the L-amino acids may be involved in a type of equilibrium reaction in initiating germination (8). The purpose of this study was to provide further information concerning the specific role of amino acids in the germination of B. licheniformis spores. Received for publication June 13, 1965. 1 Article no. 4-65. This investigation was supported by Public Health Service Grant No. EF00180-05 from the Division of Environmental Engineering and Food Protection, Bureau of State Service.

E X P E R I M E N T A L PROCEDURE

Previously described methods were used for the preparation and standardization of spore suspensions, measurement of germination response (7), and preparation of cellular fractions (8). Preparation of radioactive spores. Radioactive sporulation medium was prepared by adding 0.1 ~c/ml of a uniformly Cl'-labeled amino acid (Volk Radiochemieal Corporation) to 1.00 ml of nutrient agar (Difco) containing 1% soluble starch and 0.01% manganese sulfate. The p i t was adjusted to 6.8 and the medium was sterilized at 15 lb of pressure for 15 rain. A g a r slants were prepared and actively growing vegetative cells of B. licheniformis from thioglycollate broth were inoculated on to the surface. These slants were incubated at 35 C for four or five days, for sporulation to occur. The spores were then removed from the agar slants and washed six times with sterile phosphate buffer ( p H 7.2) to remove radioactive debris.

1184

Determination of free D-alanine in spore

GERMINATIO:N

extracts. The enzyme D-amino acid oxidase (hog kidney), obtained from the Nutritional Biochemicals Company, was utilized to determine the presence of free D-alanine in spore extracts. A combination of the procedures of various workers was necessary to obtain optimum activity of the enzyme (5, 6, 9). The procedure for the conversion of D-alanine to pyruvic acid was as follows: Two milliliters of the spore extract (germination exudates, cytoplasmic fractions, or the spore coats) were pipetted into a sterile screw-cap test tube. One-tenth milliliter (0.05 rag) of Flavine Adenine Dinucleotide ( F A D ) was added, based on the findings by Lippmann et al. (6) that the addition of F A D to the reaction mixture provided a threefold increase in the oxidation of D-alanine to pyruvic acid by D-amino acid oxidase. One milliliter of eatalase solution was added to the above mixture to block the degradation of pyruvic acid to acetic acid (9). The catalase solution was prepared in sterile pyrophosphate buffer, p H 8.3, in a concentration of 5 rag/milliliter. Finally, 1.0 ml of D-amino acid oxidase containing 8 mg/ml of the enzyme in pyrophosphate buffer, p H 8.3, was added, and the reaction mixture was brought to a total volume of 10 ml with the pyrophosphate buffer. Since Krebs (5) found the optimum activity of Damino acid oxidase occurred at p H 8.3, the reaction was conducted at pill 8.3. The mixture was warmed to 37 C, and incubated for ] hr. The amount of pyruvic acid formed from the D-alanine was determined eolorimetrieally by slight modification of the method outlined by Friedman and Haugen (3). The modified procedure was as follows: The spore extracts reacted with D-amino acid oxidase were cooled to 25 C in a water bath. To two milliliters of the extract was added 1 ml of 2,4-DNPH (prepared as a 0.1% solution in 2 iv HC1), the mixture shaken, and allowed to react at room temperature for 5 rain. Three milliliters of benzene were added, and the mixture shaken for 2 min to extract the colored 2,4-DNPH derivatives. One milliliter of the benzene layer was removed, 6 ml of 20% Na.~CO, added, and this mixture shaken for 2 mill. Five milliliters of the solution were placed in clean, dry test tube, 5.0 ml of 1.5 ~ NaOH added, and the solution mixed thoroughly. The optical density of the resulting solution was determined at 520 m~ within 10 min after addition of the NaOH. The amount of pyruvic acid was calculated from a standard curve prepared with known concentrations of pyruvie acid subjected to the same procedure. Chromatographic analysis of the radioactive

OF SPORES

]185

extracts. Amino acids in the spore extracts were resolved by ascending chromatography on Whatman no. 1 filter paper, using the solvent system (methanol, water, pyridine---20 :5 :1 : v/v) described by Young (]5). A total of 50 td of the radioactive extract was applied in 10-tL1 aliquots in /-in. bands. The ehromatograms were sprayed with niuhydrin (0.4% in butanol) and dried at 100 C until the characteristic blue color developed. Identification of the amino acids was accomplished by comparison of the position of the spots with those of known standards developed under identical conditions. The radioactivity of the material on the p a p e r was determined by cutting 30-ram circles of paper, starting at the point of application and continuing through the ninhydrin-positive spot representing the particular amino acid assayed. These circles of p a p e r were placed on planchets, and the radioactivity was determined as described previously (8). Identical circles of the p a p e r to which no extract was applied were subjected to the same procedure, to serve as controls. RESULTS

Nonradioactive L-alanine germination. During formation of B. licheniformis spores on nutrient agar containing radioactive L-alanine, about twice as much radioactivity was incorporated into the cytoplasm as into the spore coats, with 40 cpm/0.5 ml in the cytoplasm and 20 cpm/0.5 ml in the spore coats. These radioactive spores were germinated with nonradioactive L-alanine and the germination percentages were 0, 98.4, 98.8, 99.1, and 99.2 after incubation for 0, 30, 60, 120, and 240 rain, respectively. The distribution of radioactivity in the germination exudates, cytoplasm, and coats of these spores is illustrated in Figure 1. Changes in the spores occurred immediately upon incubation, as revealed by the presence of about 15 cpm/0.5 ml of radioactivity in the spore exudates at the zero-time. During the first 30 rain of incubation (during which time 98.4% of the spores germinated) there was a rapid release of radioactivity into the germination exudates, with approximately 75% of the radioactivity being released from the cytoplasm and 25%from the spore coats. No further release of radioactivity occurred during the remainder of the incubation period. These data, together with previous findings that D-alanine inhibited L-alanine-induced germination (7), suggested a possible interrelationship between the isomeric forms of a]anlne sawn ta ~f;---_ and the germination process. Ther.~_r~,

1186

J. tt, M A R T I N

EXUDATES

AND W. J. H A R P E R

•

E

r-.

, !20

SPORE GOAT S

CYTOPLASM

I¢

~o

~;~o

'

INCUBATION TIME

ibo

coats. These spores were germinated with nonradioactive L-valine and the germination percentages were 0, 43.2, 48.4, 52.6, and 66.8% after incubation for 0, 30, 60, 120, and 240 min, respectively. The distribution of radioactivity during Lvaline germination is illustrated in Figure 2. The release of radioactivity into the exudates increased from 20 cpm/0.5 ml at the zero-time to 38 cpm/0.5 ml and 65 cpm/0.5 ml after 30 rain and 4 hr of incubation, respectively. The radioactivity originated from both the cytoplasm and spore coats. After 4 hr of incubation, approximately 50% of the radioactivity in the exudates had come from the cytoplasm and the remaining 50% from the spore coats. The marked release of radioactivity from the spore coats is a striking difference from results obtained with nonradioactive La!anine as the germinant.

2'40

7¢

(MIN.)

EXUDATES

Distribution of radioactivity from spores labeled with radioactive L-alanine during germination induced by nonradioactive L-alanine. (Average of three trials.) FIO.

1.

lizing the enzyme D-amino acid oxidase to convert D-alanine to pyruvic acid, the free D-alanine in the exudates, cytoplasm, and spore coats was determined. The data obtained arc presented in Table 1. Free D-alanine was found only in the germination exudates and not in the cytoplasm or coats, nor in any fraction of the ungerminated spores. There were 9.9 t~g/milliliter of free D-alanine in the spore exudates immediately after the addition of nonradioactive L-alanine and 15.8 t~g/milliliter after 240 rain of incubation. Nonradioactive L-valine germination. During a second harvest of the spores on nutrient agar containing radioactive L-alaninc, 97 cpm/0.5 ml of radioactivity were incorporated into the cytoplasm and 52 cpm/0.5 ml in the spore

P1 0

& >.

_> 0 a

~2G

0

60

120

180

(MIN.}

TABLE 1 Free n-alanine in extracts from radioactive L-alanine-labeled spores germinated with nonradioactive L-alanlne a Incubation time at 35 C

240

iNCUBATION TiME F1G. 9,. Distribution of radioactivity from radioactive L-alanine-labeled spores during germination induced by nonradioactive L-valine. (Average of three trials.)

Micrograms D-alanine/milliliter in

(rain)

Percentage germination

Exudates

0 30 60 120 240 Control

0 98.4 98.8 99.1 99.2 None

9.9 12.0 12.4 13.4 15.8 None

Cytoplasm None None None None None None

Spore coats None None None :None None None

a Average of three trials. Loss of heat resistance utilized in determining germination.

GEI~lVIINATION OF SPOI%ES Free D-alanine was not detected in the germination exudates, cytoplasm, or spore coats of L-valine-germinated spores. G e r m i n a t i o u of radioactive L-valine-Iabeled spores. Spores were labeled with radioactive L-valine and germinated with nonradioactive L~alanine and L-valine. During formation of the spores, 51 epm/0.5 ml were incorporated in the cytoplasm and 41 epm/0.5 ml in the spore coats. Data obtained during germination are presented in Table 2. The distribution of radioactivity resembled that obtained with L-alaninelabeled spores. The spore exudates, cytoplasm, and spore coats were analyzed, using D-amino acid oxidase to convert D-amino acids to the corresponding keto acids. No difference in the color of the 2,4-DNPtI derivatives was detected between 5 and 30 rain of reaction with the extracts and based on the findings of Friedman and Haugen (3), that D-valine requires more than 5 min for conversion, results were calculated as D-alanine. Data obtained from analysis of the various fractions (Table 3) reveal that D-alanine was released into the germination medium when n-valine-labeled spores were germinated with nonradioactive L-alanine. The amount of free D-alanine appearing in the exudates was similar

1187

to that from the L-alanine-labeled spores, ranging from 6.2 tLg/ml at the zero-time to 15.3 ~g/ml after 4 hr of incubation. Traces of Dalanine were detected in the cytoplasm of these spores, but none was found in the spore coats. Free D-alanine was not detected in the exudates, cytoplasm, or coats of spores germinated with L-valine. S t u d i e s on the chemical nature o f the germination exudates. Exudates obtained during amino acid-induced germination of radioactive spores were analyzed for amino acids by paper chromatography and the distribution of radioactivity on the chromatograms was determined. Results are presented in Table 4. No significant radioactivity was detected on the chromatograms, except where ninhydrin-positive areas were present, and only two such areas were detected, one which was nomnobile and the other with an R f value of 0.78 (alanine). With L-alanine-labeled spores prior to germination (the zero incubation time) only slight radioactivity was present on the chromatogram, and it was about evenly distributed between the nonmobile component and the alanine spot. In contrast, after approximately 99% of the spores had germinated (the 30-rain incubation interval), radioactivity in the exudates had

TABLE 2 Distribution of radioactivity from spores labeled with radioactive L-valine during germination with nonradioactive n-alanine and L-valinc a L-alanine-germinated

Incubation time

Per cent germination

(min) 0 30 60 120 240 a Average

L-valine-germinated

Radioactivity (epm/0.5 ml)in Exudates

0 29 98.2 67 98.6 68 98.6 69 99.4 68 of three trials.

Cytoplasm 45 16 17 16 13

Coats 27 23 24 22 17

Radioactivity (cpm/0.5 ml) in

Per cent germination

Exudates

Cytoplasm

Coats

0 43.2 48.4 52.6 66.8

36 70 74 80 78

38 19 20 17 ]6

37 33 23 24 19

TABLE 3 Free D-a]anine in extracts from radioactive n-va]ine-labeled spores germinated with nonradioactive L-alanine ~ Incubation time at 35 C (~nin)

Percentage germination

0 0 30 98.2 60 98.6 ]20 99.2 240 99.4 Control None Average of three trials.

Micrograms n-alanine/milliliter in Exudates

Cytoplasm

Spore coats

6.2 10.5 ]] .3 ]2.9 15.3 Non e

Trace Trace Trace Trace None None

None None None None None None

J. It. M A R T I N AND W. J. H A R P E R

1188

TABLE 4 Radioactivity of ninhydrin-positive spots from paper chromatograms of exudates from germinating spores ~ Cpm at specific incubation times

(rain) Component

Rf value

C~4-n-alanine-labeled--C~:-L-alanine-germinated 1 0.03 2 0.78 Total ......

0

30

60

120

240

spores 4 3 7

13 36 49

20 42 62

17 32 49

13 30 43

9 20 29

13 24 37

11 25 36

18 12 30

3 4 7

5 4 9

C~4-L-valine-labeled--C~Z-L-alanine-germinated spores 1 0.03 2 2 0.78 2 Total ...... 4

C~4-L-valine-labeled--C~2-L-valine-germinated spores 1 0.03 3 4 4 2 0.78 1 3 5 Total 4 7 9 Radioactivity not corrected for self-absorption. Average of two ¢rials.

......

increased about sevenfold, to 49 cpm. Approximately three-fourths of this radioactivity was detected in the alanine area. This distribution of radioactivity remained essentially the same for the duration of the incubation period. Essentially the same trend was evident when n-valine-labeled spores were germinated with nonradioactive L-alanine. 0nly slight radioactivity (4 cpm) was present ou the chromatograin at the zero-time. However, after germination, the radioactivity increased significantly, with about 30-35 cpm being detected during the remainder of the incubation period. As with the L-alanine-labeled spores, the greater portion (66%) of the radioactivity migrated with the free alanine. In contrast to L-alanine-germinated spores, when L-valine-labeled spores were germinated with nonradioactive L-valine, the amount of radioactivity on the chromatograms was low, and about 50% of the radioactivity remained on the base line ( R f ~ 0 . 0 3 ) , irrespective of germination time. Ultraw;olet spectroscopy. To further characterize the germination exudates, ultraviolet absorption spectra were determined between 150 and 350 mt~ with a Bausch and Lomb Speetronic 505 Spectrophotometer. The UV spectra of dipicolinie acid ( D P A ) , alanine, and a 1:1 mixture of alanine and D P A were determined to provide reference spectra (Figure 3, a). The reference spectra revealed that when 1:1 mixtures of alanine and D P A were analyzed the UV spectrum was characteristic of the mixture and differed from that of the individual eomDounds. The UV absorption spectra for the germina-

tiou exudates from L-alanine-labelcd, L-alaninegerminated spores are presented in Figure 3, b. The absorption spectra of the spore exudates prior to germination (the zero-time interval) were markedly different from exudates of the germinated spores (the 30-ufin interval). Spectra obtained during further incubation were identical except for concentration differences.

(a)

= o Z

~!

n,, 0

t0c

ALANINE ALANINE + DPA DPA

250 500 WAVELENGTH(mp)

550

(b) (

A

g

/ /

/

/ / //'//~ ///

D

~ B

f

-

-

B- 30 min.

E

G-6Omin. D - 120min. E - 24.Ornin.

100

250

300

550

WAVELENGTH (mp) FIG. 3. Ultraviolet absorption spectra of known compounds (Figures 3, a) and of the exudates from I,-alanine-labeled, I,-alanine-germinated spores (Figure 3, b).

1189

GERMINATION OF SPOREs

Spectra obtained after spore germination were superimposable on the spectra Obtained with the 1:1 mixture of alanine and DPA. These data reveal that DPA was a major component of the exudates from germinated spores, but was not present in the exudates prior to germination. Similar results were obtained with L-valine-labeled spores. DISCUSSION

This study showed that radioactive amino acids or amino acid-derived materials were released into the germination exudates from spores labeled with radioactive amino acids after addition of nonradioactive amino acids as germination stimulants. This finding suggests that some type of an exchange reaction was involved between the amino acid in the spore and the amino acid used as the germinant. This exchange phenomenon occurred only during the germination process itself. Since previous work (8) showed there was also a marked uptake of the amino acids during post-germinative development, it would appear that the role of amino acids in germination and post-germinative development is completely different. The finding of D-alanine in the germination exudates of spores germinated with the L-alanine brings into focus results of Stewart and Haivorson (13), which revealed the presence of a highly active alanine racemase enzyme system in the spores and vegetative cells of several aerobic bacilli. Their feeling was that a negative feed-back t y p e of mechanism regulated spore germination, in which L-alanine was continuously being converted to D-alanine by this enzyme in the dormant spore. Since D-alanine has been shown to be a very effective germination inhibitor, the conversion of L- to D-alanine appeared to be the survival mechanism for bacterial spores. However, Church et al. (2) later concluded that spore germination was independent from alanine racenmse activity. Present results indicate that regulation of spore germination could depend, at least in part, on a-lanine racemase activity. Data obtained in this study suggest that the chief function of alanine racemase is in the vegetative cell during spore formation, rather than in the dormant spore itself. Conceivably, the alanine racemase of the vegetative cell actively converted L-alanine to D-alanine during formation of the spore, finally reaching a point where the spore was practically void of the L-form. The spore then remained in the dormant state because of the inhibitory effect which D-alanine exerted on the germination process, until such time that sufficient L-alanine was introduced to reverse

the ratio of D- to n- to a level favorable for spore germination. The study of the relationship between D-alanine and L-alanine revealed that D-alanine in the free state could not be detected in the cytoplasmic fraction or in the spore coats at any time during germination and subsequent post-germinative development. This suggests that D-alanine exists in the spore in a bound or complexed form. Several possibilities as to the form in which the amino acid exists in the spore are a) as a part of an acid-soluble nucleotide complex, b) as some type of amino acidDPA-chelate complex, such as that suggested by Riemann (11) and Young (15), or e) the amino acid could be complexed with some structure or structures within the spore, such as the teiehoie acids reportedly found in many other microorganisms (1, 4). The ultraviolet absorption studies which revealed that dipicolinic acid was released from the spores into the surrounding medium after germination lend support to the suggestion by Young (15) that alanine is associated with the dipicolinic acid released from spores. However, in this study the rates of release of alanine and dipicolinic acid from spores differed markedly, with D-alanine being detected in the exudates almost immediately after initiation of the germination process and dipieolinic acid appearing in the germination exudates only after the majority of the spores had germinated. I f the alanine is complexed with dipicolinic acid in the intact spore, then the complex must be broken inside the spore, and this might be considered the trigger mechanism. REFERENCES

(]) AX~ST~ONO,J. J., BADmLEY,J., BVC~ANAN, J. G., CAESS, B., AND GREENBERG, G. R.

1958. Isolation and Structure of Ribitol Phosphate Derivatives (Teichoie Acids) from Bacterial Cell Walls. J. Chem. Soc., 4344. (2) C~tURCtt, B. D., HALVORSON, H., AND HAL-

VORSON, It. O. 1954. Studies on Spore Germination: Its Independence from Alanine Racemase Activity. J. Bacteriol., 68: 393. (3) FRIEDMAN, T. E., AND HAUGEN, G. E. 1943. Pyruvic Acid. II. The Determination of Ke~o Acids in Blood and Urine. J. Biol. Chem., 147 : 415. (4) IKAWA, M., AND SNELL, B. E. 1960. Cell Wall Composition of Lactic Acid Bacteria. J. Biol. Chem., 235: 1376. (5) K~EBS, tI. A. 1935. Metabolism of Amino Acids. III. Deamination of Amino Acids. Biochem. J., 29: 1620.

(6) I,IPP]PIANI'JyF., ~-/OTCI-IKISS,R. D., AND DUBAg,

1190

J. H. MA}tTIN AND W. J. HARPER

R. 5. 1941. The Occurrence of ])-amino Acids in Gramieidin and Tyrocidine. J. Biol. Chem., 141: 163. (7) MARTIN, J. ILl., AND tIARPER, W. J. 1963. Germinatiott Response of B a c ~ s ~ieheuiformis Spores to Amino Acids. J. Dairy Sci., 46 : 663. (8) MARTIN, J. H., AND HAI~PEI¢, W. J. 1965. The Role of Amino Acids in the Germination of Bacillus licheniforcnis Spores. I. Uptake of Radioactive Anfino Acids by Germinating and Dormant Spores. J. Dairy Sci., 48: 282. (9) NEGELEIN, ]~., AND BRO)IEL, I~. 1939. Prorein der D-amlnosaureoxydase. Biochem. Z., 300 : 225. (10) POWELL, J. F. 1957. Biochemical Changes Occurring During Spore Germination in Bacillus Species. J. Appl. Bacterio]., 20: 349.

(11) RIE-MANN,}[. 1961. Germination of BacCeria (12)

(13)

(14)

(15)

by Chelating Agents. Spores II, p. 24. Burgess Pubh Co., Minneapolis, Minn. RODE, L. J., AN1) FOSTER, J. W. 1960. The Action of Surfactants on Bacterial Spores. Arch. Mikrobiol., 36: 67. STEWART, B. T., AND HALVO~SON, H. O. 1953. Studies on the Spores of Aerobic Bacteria. I. The Occurrence of Alanine Racemase. J. Bacteriol., 65: ]60. ST~ANOE, R. E., ANP POWELL, J. F. 1954. Hexosamine-containing Peptides in Spores of Bacillus subtilis, Bacillus ¢negaterium, and Bacillus cereus. Biochem. J., 58: 80. YOUNg, I. E. 1959. A Relationship Between the Free Amino Acid Pool, Dipicolinic Acid, and Calcium fronl Resting Spores of Bacillus megaterium. Canadian J. Microbiol., 5 : ]97.