STREPTOCOCCUS CREMORIS'

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1 IDENTIFICATION OF STIMULATORY FACTOR INVOLVED IN SYMBIOTIC GROWTH OF STREPTOCOCCUS LACTIS AND STREPTOCOCCUS CREMORIS' R. S. DAHIYA AND M. L. SPECK Department of Food Science, North Carolina State College, Raleigh, North Carolina Received for publication 6 October 1962 ABSTRACT to grow independently. However, the associative DAHIYA, R. S. (North Carolina State College, growth resulted in much-enhanced acid production over that produced by the single-strain Raleigh) AND M. L. SPECK. Identification of stimulatory factor involved in symbiotic growth cultures. Some of the characteristics of the of Streptococcus lactis and Streptococcus cremoris. stimulatory factor(s) involved in a symbiotic J. Bacteriol. 85: Single-strain cultures of Streptococcus lactis and S. cremoris iso- during growth in milk were studied by Dahiya relationship among strains of lactic streptococci lated from a commercial starter culture showed and Speck (1962). The present study deals with symbiotic growth in milk. This study dealt with the isolation and identification of the factor from identification of the main component responsible Streptococcus lactis which provided the main for high acid production resulting from the combined growth of these cultures. Paper chroma- between it and S. cremoris. source of stimulation in the symbiotic relationship tography and bioautography were adapted to MATERIALS AND METHODS isolate the main stimulatory factor excreted by the slower-growing culture (S. lactis) in the The isolation and maintenance of the streptococci used in the present study were described culture media. It was identified as adenine on the basis of ultraviolet absorption spectra and paper earlier (Dahiya and Speck, 1962). S. lactis FIA chromatographic RF value. Pure adenine possessed the same properties and was also stimula- about 72 hr at 22 C to produce sufficient acid for grew relatively slowly in litmus milk, requiring tory to the faster-growing culture (S. cremoris) coagulation; S. cremoris F2A was a faster-growing when added to milk. culture that acid-coagulated litmus milk in 24 hr at 22 C. Preparation of filtrate from milk cultures of these strains, the adsorption and elution of Various studies have shown that different the active material from a resin column (Amberlite IR 120, H+ cycle), the hydrolysis of different strains of lactic acid bacteria could be grown symbiotically in a synthetic medium that was culture fractions, and the method used for tube incapable of supporting the growth of either assay have also been described previously organism in pure culture. The results of these (Dahiya and Speck, 1962). studies pointed out that each strain produced a Removal of free amino acids by ninhydrin growth factor required by the other, hence treatment. A modification of the method of supplying each other's needs when grown in Markovitz and Steinberg (1957) was used. To a association (Nurmikko, 1953; Doctor and Couch, portion of the resin eluate from the ion exchange 1954; Koft and Morrison, 1956). Associative column, citric acid was added to make the solution ph 2.2 to 2.3, and 0.1 N NaOH was added growth among lactic streptococci in milk observed by Greene (1959), Olson (1960), and to adjust the material back to ph 2.5. Ninhydrin Dahiya and Speck (1962) presented a somewhat (0.06 g/ml of resin eluate) was then mixed with different situation, since the cultures were able the eluate. The tube containing the mixture was 1 Contribution from the Department of Food placed in boiling water for 15 min with continued Science, North Carolina Agricultural Experiment Station, Raleigh. Published with the ap- and was removed by filtration. The treated stirring. During the heating, a precipitate formed proval of the Director of Research as paper no. filtrate was cooled in an ice-water bath. The 1517 of the Journal Series. solution was extracted five times with ether to 585

2 586 DAHIYA AND SPECK J. BACTERIOL. TABLE 1. Effects of different treatments of column eluate (from Streptococcus lactis FIA) on stimulation of S. cremoris F2A Sample* Incubationt (at 22 C) for 16hr 18hr Control (F2A) Resin eluate (milk only) Resin eluate (FlA) Hydrolyzed resin eluate (FlA) Ninhydrin-treated resin eluate (FlA) Hydrolyzed ninhydrin-treated resin eluate (FlA) * Each addition added in 1% concentration (based on volume of original filtrate). t Results expressed as ml of 0.1 N NaOH per 10 ml of culture. remove any unreacted ninhydrin. The ether was removed by condensing under vacuum. The aqueous layer was again passed through the resin column. After washing the column with distilled water, the material was eluted out with 4% aqueous ammonia solution. The ammonia was removed by repeated vacuum condensing after adding water. Paper chromatography. One-dimensional descending paper chromatography was performed at room temperature. Paper strips from rolls of Whatman no. 3 MM chromatographic paper were used. The following solvent systems were used for development: (A) butanol-methyl ethyl ketone-water (2:2:1), as recommended by Mizell and Simpson (1961); and (B) butanol-acetic acidwater (50:6:50), as recommended by Woiwod (1949). The strips were developed for 20 hr during the isolation of the stimulatory factor. The solvent front ran off the bottom of the strips during this time, and the zones observed on these chromatograms, either after developing with ninhydrin solution (Tonnies and Kolb, 1951) or by scanning with ultraviolet light (254 m,u wavelength), were indicated by the distances they traveled from the base line. RF values were determined in the usual manner, and the strips for these purposes were developed for only 10 hr so that the solvent front could be marked near the end of the strips. Bioautographic assay. The bioautographic technique of Speck, McAnelly, and Wilbur (1958) was used in this study to locate areas of biological activity on paper chromatograms. RESULTS Earlier studies (Dahiya and Speck, 1962) showed that S. lactis (FlA) produced a certain compound(s) while growing in milk, which was stimulatory for S. cremoris (F2A). The compound involved was presumed to be of row molecular weight, as it was lost on dialysis through cellophane tubing. Further observations on the nature of the compound are recorded in Table 1. The data indicated that a compound(s) stable to acid hydrolysis, such as an amino acid(s), might be contributing to the major part of the activity in the culture filtrate, and that a minor part of the activity might be associated with a heat-labile peptidelike compound. Subsequently, a portion of the eluate from the resin column was treated with ninhydrin to remove the free amino acids. The ninhydrintreated eluate, before and after acid hydrolysis, was tested for stimulation of S. cremoris F2A. The results (Table 1) indicated that a major part of the activity was lost during ninhydrin treatment of resin eluate. This observation further suggested that a compound(s), like an amino acid(s) or an amine(s) with a free amino group, was mainly responsible for stimulation of S. cremoris F2A. Compounds of these types would be expected to be stable to acid hydrolysis and to be removed during ninhydrin treatment. The minor part of the activity in the ninhydrintreated eluate that was lost on acid hydrolysis suggested that a peptide might be involved. To isolate the major stimulatory factor, paper chromatograms of resin eluate and hydrolyzed resin eluate were prepared by use of butanolmethyl ethyl ketone-water (2:2:1). These chromatograms were then tested on litmus milk agar bioautographic plates to locate the zone(s) active for S. cremoris F2A. The bioautographic plates, with a chromatogram of resin eluate and a chromatogram of hydrolyzed resin eluate, consistently showed one distinct stimulatory zone (33.4 cm from the base line). This zone always coincided with the ultraviolet-absorbing zone, but it did not react with ninhydrin. To characterize the compound in the active zone, it was eluted from a number of chromatograms, and the ultraviolet-absorption spectra at ph 1.0 and 14.0 were determined. From consultation of available

3 VOL. 85, 1963 STIMULATORY FACTOR FOR S. LACTIS AND S. CREMORIS TABLE 2. Comparison of compound excreted by Streptococcus lactis FlA with adenine Test Compound Adenine excreted Chromatography (RF) Solvent A Solvent B Ultraviolet absorption (m,) at ph 1.0 Maximum Minimum Optical density, 250/ Optical density, 280/ Ultraviolet absorption (m,u) at ph 14.0 Maximum Minimum Optical density, 250/ Optical density, 280/ TABLE 3. Stimulation of Streptococcus cremorts F2A by eluate (from S. lactis F1A) of active zone on paper chromatograms and known adenine Sample identification Amt Amt of eluate added 0.1% 1% 4% jag/mi Eluate-active zone Hydrolyzed eluate active zone Adenine Control (F2A) * Results expressed as ml of 0.1 N NaOH per 10 ml of culture. Incubation at 22 C for 18 hr. literature (Beaven, Holiday, and Johnson, 1955), the compound involved was suspected of being adenine. To confirm this, the ultraviolet-absorption spectrum of the known adenine solution was also determined. Furthermore, the RF value of the eluate from the active zone and the known adenine solution were also compared on paper chromatograms developed in two different solvent systems. The results (Table 2) confirmed the earlier observation that the compound present on the active zone was adenine. The next step was to determine whether adenine was the same stimulatory compound present in the resin eluate earlier (Table 1) observed to be removed during ninhydrin treatment. For determining this, the known adenine solution was treated with ninhydrin in the manner described earlier for resin eluate. No active zone was observed when a paper chromatogram, prepared from this ninhydrin-treated adenine solution, was tested by bioautograph. This indicated that the ninhydrin treatment of the eluate removed adenine, as well as amino acids, and explained the loss of activity (Table 1) by this treatment of the resin eluate. Finally, both the eluate from the active zone from paper chromatograms and the known adenine solution were assayed in milk for stimulation of S. cremoris F2A. The results (Table 3) indicated that the eluate from the active zone, as well as known adenine, stimulated the growth of this culture. By increasing the concentration of the eluate, it was possible to obtain as much stimulation as was obtained by the addition of adenine. The results further confirmed that the main stimulatory compound excreted by culture S. lactis FlA was adenine. DISCUSSION This investigation demonstrated that the excretion of adenine in the culture media (milk) by the growth of S. lactis FlA was the main factor responsible for high acid production by the mixed culture of S. lactis FlA and S. cremoris F2A. The metabolic phenomenon involved in the accumulation of adenine in the S. lactis FlA culture can only be speculated at the present stage of study. One explanation seems to be that an active hydrolytic enzyme is present in the cells which hydrolyzes adenosine or adenylic acid at a rate faster than it was being incorporated into compounds such as ribonucleic acid, diphosphopyridine nucleotide (DPN), adenosine triphosphate (ATP), etc. The adenine thus produced was being leached out of the cells and was accumulating in the culture medium. Hydrolytic nucleosidase which catalyzed the hydrolysis of uridine was found in yeast plasmolysates by Carter (1951). Lampen and Wang (1952) observed the presence of purine and pyrimidine nucleoside hydrolyase in Lactobacillus pentosus and indicated hydrolytic cleavage as an important

4 588 DAHIYA AND SPECK J. BACTERIOL. pathway of purine and pyrimidine riboside degradation in this organism. Further, it seems likely that the salvage mechanism for reutilizing adenine for growth via adenylic phosphorylase is absent in S. lactis FlA. Addition of known adenine in the culture medium had no effect on the growth of this organism. Recently, Kalle and Gots (1962) also observed excretion of adenine in culture media by a mutant strain of Salmonella typhimurium. The studies on mutants that excrete metabolites led to the general belief that in all of these instances the over-production was due to some defect in the mechanism involved in control of biosynthesis of these metabolites (Cohen and Adelberg, 1958; Adelberg, 1958; Moyed and Friedman, 1959; Moyed, 1960). However, Kalle and Gots (1962) observed that the mechanism for control of purine biosynthesis by feedback inhibition was unaffected in the mutant strain that excreted adenine, and suggested that a mechanism other than a mere defect in control of purine biosynthesis might be involved. The results further indicate that S. cremoris F2A was unable to synthesize adenine in the required amount for optimal growth, and that addition of this compound to the culture media was stimulatory for its growth. Since adenine is a component of nucleic acid, it is reasonable to assume that it is used by the organism to synthesize certain essential protoplasmic units. However, this may not be the only function, since certain known coenzymes (ATP, DPN, coenzyme A) active in various phases of hexose metabolism contain this ring structure as part of the molecule. Adenine has been observed to stimulate the growth of certain strains of lactic streptococci in synthetic media. Rahn and Hegarty (1938) reported that adenine might increase the lactic fermentation of S. lactis in combination with other factors, but not by itself. Snell and Mitchell (1941) noted that adenine stimulated the growth of and appeared to be essential for S. lactis, and that, to obtain maximal growth, adenine, guanine, thymine, or uracil must be included in the synthetic media. Anderson and Elliker (1953) observed that, of 32 strains of lactic streptococci isolated, adenine was essential for 4 strains and stimulated the growth of 7 other strains. The results obtained in the present study point out that better understanding of the capabilities for synthesis of certain growth factors by the lactic streptococci would aid measurably in explaining some of the symbiotic relationships of these bacteria. ACKNOWLEDGMENT This study was supported in part by a grant from the American Dairy Association. LITERATURE CITED ADELBERG, E. A Selection of bacterial mutants which excrete antagonists of antimetabolites. J. Bacteriol. 76:326. ANDERSON, A. W., AND P. R. ELLIKER The nutritional requirements of lactic streptococci isolated from starter cultures. I. Growth in a synthetic medium. J. Dairy Sci. 36: BEAVEN, G. H., E. R. HOLIDAY, AND E. A. JOHN- SON Optical properties of nucleic acid and their components, p In E. Chargaff and J. N. Davidson [ed.], The nucleic acids. Academic Press, Inc., New York. CARTER, C. E Partial purification of nonphosphorylytic uridine nucleosidase from yeast. J. Am. Chem. Soc. 73: COHEN, G. N., AND E. A. ADELBERG Kinetics of incorporation of p-fluorophenylalanine by a mutant of Escherichia coli resistant to this analogue. J. Bacteriol. 76: DAHIYA, R. S., AND M. L. SPECK Symbiosis among lactic streptococci. J. Dairy Sci. 45: DOCTOR, V. M., AND J. R. COUCH An unusual example of symbiosis in bacteria. Arch. Biochem. Biophys. 51: GREENE, V. W Interaction among pure strains of lactic streptococci isolated from multiple strain cheese starters. J. Dairy Sci. 42:906. KALLE, G. P., AND J. S. GOTS Metabolic abnormalities of 2,6-diaminopurine-resistant mutants of Salmonella typhimurium. Proc. Soc. Exptl. Biol. Med. 109: KOFT, B. W., AND J. H. MORRISON Symbiotic biosynthesis of folic acid-like growth factors. J. Bacteriol. 72: LAMPEN, J. O., AND T. P. WANG The mechanism of action of Lactobacillus pentosus nucleosidase. J. Biol. Chem. 198: MARKOVITZ, A., AND D. STEINBERG A method for the determination of peptides in the presence of free amino acids. J. Biol. Chem. 228: MIZELL, M., AND S. B. SIMPSON, JR Paper chromatographic separation of amino acids. A solvent to replace phenol. J. Chromatog. 5:

5 VOL. 85, 1963 STIMULATORY FACTOR FOR S. LACTIS AND S. CREMORIS 589 MOYED, H. S False feed-back inhibition: inhibition of tryptophan biosynthesis by 5-methyl tryptophan. J. Biol. Chem. 235: MOYED, H. S., AND M. FRIEDMAN Interference with feed-back control: a mechanism of antimetabolite action. Science 129: NURMIKKO, V Symbiosis among microorganisms as related to growth factor requirement. Intern. Congr. Microbiol., 6th, Rome, p OLSON, H. C Symbiosis in lactic cultures. J. Dairy Sci. 43:439. RAHN, O., AND C. P. HEGARTY Accessory factors in lactic fermentation. Proc. Soc. Exptl. Biol. Med. 38: SNELL, E. E., AND H. K. MITCHELL Purine and pyrimidine bases as growth substances of lactic acid bacteria. Proc. Natl. Acad. Sci. U.S. 27:1-7. SPECK, M. L., J. K. MCANELLY, AND J. D. WILBUR Variability in response of lactic streptococci to stimulants in extracts of pancreas, liver and yeast. J. Dairy Sci. 41: TONNIES, G., AND J. KOLB Techniques and reagents for paper chromatography. Anal. Chem. 23: WOIWOD, A. J A technique for examining large number of bacterial culture filtrates by partition chromatography. J. Gen. Microbiol. 3: