STARCH HYDROLYSIS BY STREPTOCOCCUS EQUINUS

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1 STARCH HYDROLYSIS BY STREPTOCOCCUS EQUINUS LAWRENCE K. DUNICAN' AND HARRY W. SEELEY Division of Bacteriology, Department of Dairy and Food Science, College of Agriculture, Cornell University, Ithaca, Ne York Received for publication July 21, 1961 ABSTRACT DUNICAN, LAWRENCE K. (Cornell University, Ithaca, N. Y.) AND HARRY W. SEELEY. Starch hydrolysis by Streptococcus equinus. J. Bacteriol. 82: In a study of starch hydrolysis by strains of Streptococcus equinus, 52 isolates ere obtained and their amylolytic abilities determined. It as found that all the strains could hydrolyze starch to some extent hen gron in the presence of an easily fermentable carbohydrate, viz., glucose. Without this carbohydrate the organisms did not hydrolyze starch. The hydrolysis of starch as inhibited hen the organisms ere gron in an atmosphere of 5% CO2 and 95% N2, even if gron in the presence of a fermentable monosaccharide. S. bovis, hich as used as a reference organism, readily hydrolyzed starch in the absence of monosaccharides and in atmospheres containing CO2. In no instance did S. equinus hydrolyze the starch to the level of reducing sugars. Negligible amounts of reducing sugars ere recovered hen the cell-free filtrates of S. equinus ere incubated ith starch. With S. bovis, the yield of reducing sugars under such conditions as almost quantitative. These facts extend further the differences beteen these related orgaiiisms. The ability to synthesize an internal starchlike polysaccharide as noted in most of the strains of S. equinus. Synthesis as found hen the organisms ere gron on maltose or on a starch medium containing a small amount of fermentable monosaccharide. The systematic application of the starch hydrolysis test in the classification of streptococci as first used by Andrees (1930) to distinguish beteen hemolytic and nonhemolytic I Holder of an American Grant Counterpart Fund Scholarship, aarded by University College, Dublin, Ireland. species from human infections. Later, Sherman and Stark (1931) found this characteristic to be of special importance in the identification of Streptococcus bovis, hich actively hydrolyzes starch. This physiological test as used routinely in the classification of the streptococei hen Sherman (1937) reported that S. bovis as very active in hydrolyzing starch, hile S. equinus could do so only "under favorable conditions". Since that time no investigation has been undertaken to study the conditions under hich S. equinus may hydrolyze starch. S. thermophilus has also been reported to hydrolyze starch "on a favorable medium" (Breed, Murray, and Smith, 1957). Although S. equinus has remained the forgotten member of the genus, its close relative S. bovis has received ide attention due to (i) its occurrence in large numbers in the rumina of cattle and sheep (Mann and Oxford, 1955), (ii) its part in the synthesis of iodophilic polysaccharides (Hobson and Mann, 1955), (iii) its ability to synthesize a dextran from sucrose (Dain, Neal, and Seeley, 1956), (iv) the report that ammonium chloride could serve as the sole source of nitrogen for its groth (Wolin, Manning, and Nelson, 1959), and (v) reports that the organism could gro on a very simplified medium (Niven, Washburn, and White, 1948; Oxford, 1958; Barnes, Seeley, and VanDemark, 1961). Seeley and Dain (1960) made the observation that streptococci fitting the description of S. bovis could be isolated from a ide range of ruminants and ere consistent in their ability to hydrolyze starch. With this in mind, a re-examination of the hydrolytic ability of S. equinus on starch media as undertaken to see if this physiological character as of taxonomic importance in the delimitation of the to species. The observation that S. equinus had the ability to form an internal starchlike polysaccharide added to the interest in the species and made a study of the starch-splitting capabilities of these to streptococci more pertinent. 264 Donloaded from on September 12, 2018 by guest

2 1962] STARCH HYDROLYSIS BY S. EQUINUS 265 MATERIALS AND METHODS Cultures. All the strains of S. equinus described in this investigation ere freshly isolated from horse feces. Those of S. bovis and other reference streptococci used ere from the stocks of the Division of Bacteriology, Cornell University. Isolation procedure. Suspensions of fresh horse feces ere made in sterile ater (1 g/ml), and these ere streaked on a blood agar medium hich had the folloing composition: tryptone, 1.0%; meat infusion, 12 vol; NaCl, 0.5%; agar, 1.5% (ph 7A). After sterilization of the medium at 15 lb for 15 min and cooling to 42 C, hole citrated horse blood as added to a concentration of 5%. Plates ere streaked ith the manure suspensions and incubated for 2 days at 37 C. During this incubation period the plates ere examined for the presence of minute colonies shoing zones of "greening." Colonies shoing these characteristics ere isolated into broth ith the folloing percentage composition: tryptone, 1.0; yeast extract, 0.5; glucose, 0.05; K2HPO4, 0.2 (ph 7.0). Organisms conforming to the species description of S. equinus in Bergey's Manual of Determinative Bacteriology (7th ed.) ere used in the study. In this manner, 52 isolates of S. equinus, from the feces of 40 horses, ere obtained. Hydrolysis procedures. Starch hydrolysis as studied initially by folloing the disappearance of starch from starch plates. The medium used as of the folloing percentage composition: tryptone, 1.0; yeast extract, 0.5; K2HPO4, 0.2; starch (Lintner's soluble), 0.3; glucose, 0.05; agar, 1.5 (ph 7.0 to 7.2). Streak plates ere incubated for 3 days at 37 C, after hich the disappearance of starch as noted by flooding the plates ith Gram's iodine. A positive result as denoted by the absence of the usual blueblack color of the starch-iodine complex in the zones surrounding the colonies. Atmospheres of 5% CO2 and 95% N2 ere achieved by replacing the air in desiccators ith this gas mixture. The temperature of incubation in all experiments as 37 C. Utilization of starch for groth as denoted by acid production in tubes ith starch alone or starch plus glucose as metabolites. The indicator used as bromeresol purple (32 mg in 1 ml of ater and 1 ml of ethanol) at a concentration of 2 ml per liter. The amount of starch-splitting enzyme in the cell-free filtrate as determined by groing the organisms in media of the folloing percentage composition: tryptone, 1.0; yeast extr'act, 0.5; K2HPO4, 0.2; maltose, 2.0 (ph 7.2). Quantities of 400 ml of the media ere used in 500-ml flasks to give a lo oxygen tension and in 4-liter flasks on a shaker to give conditions of high -oxygen tension; 5 ml of a 24-hr culture of the to organisms ere used as inocula. The flasks ere incubated at 37 C. Samples (15 ml) ere taken at 2-hr intervals and turbidimetrically examined for groth on a Kromatrol densitometer and for ph ith a Beckman G glass-electrode ph meter. The degree to hich amylase had developed at each of the sampling intervals as found by the folloing method: The samples ere centrifuged at 8000 X g for 15 min to remove the cells. The cell-free filtrate as taken off and 1-ml quantities ere placed in each of three tubes. The filtrate in the first tube as inactivated by heating briefly at 90 C. This tube served as a control to correct for the amount of reducing sugar already present in the filtrate. The second tube as heated to 70 C for 15 min. A third tube in each set as unheated. To each tube as added 50 mg of starch, and the contents ere incubated for 3 hr. Then all the tubes ere heated to inactivate the enzymes, and the amount of reducing sugar in each as determined using the method of Somogyi (1945). The increased reducing sugars in the second and third tube over that of the control tube indicated the amylolytic activity of the filtrate. The sample heated to 70 C for 15 min as included, since it as found by Schimmer and Balls (1949) that this temperature as critical for the amylases of malt, a-amylase surviving this treatment hile,b-amylase could not. It as noticed that some of the colonies stained a blue-black color in the presence of the iodine used to demonstrate hydrolysis. An iodophilic reaction for S. equinus has not been reported previously, even though such a phenomenon is common among organisms of rumen origin, including streptococci (Smith and Baker, 1944; Doetsch et al., 1953; Robinson et al., 1955; MacPherson, 1953; Mann and Oxford, 1955). The iodine-staining material as formed hen S. equinus as gron on 2% maltose. Based on this information, all the strains of S. equinus available ere examined for their ability to store this material. The medium used had the percentage composition: tryptone, 1.0; yeast ex- Donloaded from on September 12, 2018 by guest

3 266 DUNICAN AND SEELEY [VOL. 83 tract, 0.5; K2HPO4, 0.2; maltose, 2.0 (ph 6.8 to 7.0). The tubes ere incubated for 24 hr at 37 C, after hich the organisms ere centrifuged don in a clinical centrifuge and ashed in 10 ml of ater. Gram's iodine (0.5 ml) as added to each tube, and the sediment as observed for the formation of a blue-black color indicating the presence of iodophilic material in the cells. Cells gron on the basal medium containing 1.0% glucose ere used in an effort to see if resting cells of S. equinus could synthesize the intracellular starchlike material. The cells ere gron for 24 hr, ashed in ater, and suspended in 2.0% maltose buffered at ph 5.3 and 6.7. The suspensions ere incubated at 37 C, and ere examined at hourly intervals for the accumulation of iodophilic polysaccharide. RESULTS AND DISCUSSION The results in Table 1 sho that the majority of the strains of S. equinus used in the test could hydrolyze starch under the experimental conditions used. No strain as found to be incapable of breaking don the starch hen the starch plates ere incubated in an atmosphere of air. Under the atmosphere comprised of 5% CO2 and 95% N2, no strain hydrolyzed starch. This gas mixture as used in vie of the fact that increased CO2 tension often influences the outcome of metabolic studies of streptococci. The starch-agar used in the experiments reported in Table 1 contained 0.05% glucose, added to ensure that the organism could become established in the medium. Table 2 shos that neither groth nor starch hydrolysis by S. equinus occurred hen glucose as omitted from the medium. To representative strains of S. bovis gron under the same conditions gre and hydrolyzed starch in the absence of added glucose. This seemed to indicate that S. equinus as unable to liberate glucose from starch hydrolysis in sufficient quantity to cause the initiation of groth. Alternately, the possibility existed that the hydrolyzed starch played little or no part in the metabolism of the organism. This latter hypothesis as examined by studying the production of acid from starch by S. equinus. Table 3 shos that S. equinus did not use the starch as a metabolite to any appreciable extent. The amount of glucose available in the medium as insufficient to cause any change in the color of the indicator. Acid production in a starchglucose medium, under these conditions, ould TABLE 1. Hydrolysis of starch in glucose-starch agar by Streptococcus equinus Number of Hyrlssi.i Hydrolysis in strains Hydrolysis in air 5% CO2/95% N * * Ratings: + ++ = very good hydrolysis; ++ = good; + = poor; - = no hydrolysis. TABLE 2. Hydrolysis of starch by Streptococcus equinus and S. bovis in the absence of added glucose Strains Hydrolysis in air 5% Hydrolysis CO2/95% in N2 S. equinus * (10 strains) S. bovis + + (2 strains) * Ratings: + = hydrolysis;- = no hydrolysis. TABLE 3. Production of acid from starch by Streptococcus equinus and S. bovis gron in liquid culture Strain Substrate Glucose-starch' Starch alone Glucose S. equinus 1/A +t 1/B - - _ 7/B _ 8/A _ 12/A + 14/A _ 17/B _ /B _ 38/B + S. bovis _ _ _ _ * Glucose as used at concentration. 0.05%7, starch at 0.3% t Ratings: +++ = complete change of indicator; + = slight change in color of indicator; - = no acid. Donloaded from on September 12, 2018 by guest

4 1962] STARCH HYDROLYSIS BY S. EQUINUS 267 FIG. cr 0 Cl) 0 I- -J z a. mean that the excess acid produced to change the indicator ould come from the metabolized starch. With S. bovis, on the other hand, it as found that acid as produced in the starch broth both in the presence and in the absence of the added glucose. These results indicate that the starch is not being broken don to the level of reducing sugars by S. equinus. The iodine tests used to indicate starch hydrolysis on plates actually indicate only that the starch molecules ere broken sufficiently to cause failure of formation of the starch-iodine complex. It had been suggested by Hobson and Mac- Pherson (1952) that an amylolytic streptococcus (probably S. bovis) from the rumen of sheep split starch by a constitutive rather than an induced enzyme. Klein (personal communication), orking ith Pseudomonas saccharophilia, shoed that this organism had an inducible starch-splitting enzyme hen gron on maltose. Experia 251- c ph I- z a -j( 0.1 ' k> a. OPTICAL 3 g7 DENSITY I H O U R S HEATED TO 70OC _ a. a r~~~~~~~~~~~~,t-o I -, EL 2 4 H O U R S ANAEROBICALLY GROWN CELLS AEROBICALLY GROWN CELLS 1. Production of reducing sugars from starch by cell-free filtrates of Streptococcus equinus ph ANAEROBICALLY GROWN CELLS 7 lr~ ~ ~~~~ AEROBICALLY GROWN Z6 CELLS.S 0n 100 r ments shoed that both species of streptococci under study in the present ork had the ability to produce a starch-splitting enzyme hen gron on maltose in the absence of starch. This fact made it possible to measure the amount of enzyme in the cell-free filtrates of these to organisms ithout the complication of having unused starch in the filtrate. Figures 1 and 2 sho the result of this experiment. The amount of reducing sugars recovered from the starch as very high in the filtrate from the culture of S. bovis (80%), hereas the amount in the filtrate of the culture of S. equinus as only about 10%. Maximal production of the extracellular enzyme in S. bovis occurred toards the end of the logarithmic phase of groth, and this as folloed by a decrease in activity hich corresponded closely to the ph decrease. The small activity of S. equinus reached a peak after 4 hr and then declined. The recovery of reducing sugars in cul- -J a - W-MOT Donloaded from on September 12, 2018 by guest OA ~~~~~~~. HEA'TED 'o FIG H O U R S Production of reducing sugars from starch by cell-free filtrates of Streptococcus bovis

5 268 DUNICAN AND SEELEY VOL. 83] TABLE 4. Synthesis of intracellular starch by strains of Streptococcus equinus Number of strains Reaction ith iodine * None * Ratings: = very + ++ = dark sediment; ++ = + = yello sediment. black sediment; bron sediment; tures of S. bovtis as diminished hen the filtrate as heated to 70 C for 15 min. Such a result as not found ith S. equinus. The fact that the starch-splitting enzyme of S. bovis cannot survive 70 C for 15 min makes this enzyme different from the a-amylase of barley, hich can survive this treatment (Schimmer and Balls, 1949). Hoever, it seems from this ork and that of Hobson and Mac- Pherson (1952) that the starch-splitting enzyme in S. bovis is of the alpha type. On the other hand, the enzyme-splitting starch in S. equinus is difficult to classify. It shos no similarity to the action of f3-amylase described by Neufeld and Hassid (1955), but shos in a general ay the appearance of being an a-amylase in that starch as broken don only to the level of oligosaccharides and not to reducing sugars as is generally the case. In this respect, the enzyme from S. equinus seems to resemble the nonsaccharifying type of a-amylase in Bacillus subtilis described by Kneen and Breckford (1946), hile the enzyme of S. bovis has the properties of the saccharifying a-amylase of B. subtilis. The observation that S. equinus as very active in its ability to synthesize an intracellular starchlike polysaccharide as investigated to see if it had any connection Nvith the breakdon of the starch. Table 4 shos the results hen 45 strains of S. equinus ere gron on 2% maltose and the centrifuged cells ere tested for their reaction ith iodine. Of the 45 strains examined, 27 ere found to store enough starch to give a bron to black color ith iodine. Synthesis of the polysaccharide did not occur hen the cells ere gron on glucose, fructose, or starch. The cells did not gro on the starch medium unless there as a small TABLE 5. Synthesis of intracellular starch from maltose by resting cells of Streptococcus equinus gron on glucose time ph Anaerobic Aerobic hr * _ _ * Ratings: + = synthesis; - = no synthesis. quantity of glucose or fructose present. When a small amount of these monosaccharides as present in the starch medium, starch as stored in the cells. When cells of S. equinus ere gron on a glucose medium, ashed, and suspended in buffered maltose (Table 5), it as found that an iodophilic polysaccharide as formed after 2 hr by resting cells hich had been previously gron under aerobic conditions, hereas the time interval for the polysaccharide synthesis using cells hich had been gron anaerobically as 4 hr. There as no difference in time of synthesis among the suspensions buffered at ph 6.7 and 5.3. It ould seem that the internal polysaccharide synthesized by S. equinus is formed in the same manner as that originally described by Monod and Torriani (1950) in Escherichia coli. This synthesis, ascribed to an enzyme amylomaltase, has been reported in S. pyogenes (Croley, 1955) and in group D a-hemolytic streptococei (Hobson and Mann, 1955). In both cases, the synthesis occurred only from maltose or from short-chain polymers of glucose. Croley found that internal starch synthesis in S. pyogenes as greatest in the presence of plasma and most efficient in the amylolytic strains. Such a correlation beteen the ability to form intracellular starch and amylolytic activity as also seen to a lesser extent in S. equinus. LITERATURE CITED ANDREWES, F. W Note on the fermentation of starch by certain haemolytic streptococci. J. Pathol. Bacteriol. 33: BARNES, I. J., H. W. SEELEY, AND P. J. VANDEMARK The nutrition of Strepto- Donloaded from on September 12, 2018 by guest

6 19621 STARCH HYDROLYSIS BY S. EQUINUS 269 coccus bovis in relation to dextran formation. J. Bacteriol. 82: BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH Bergey's manual of determinative bacteriology. 7th ed., The Williams and Wilkins Co., Baltimore. CROWLEY, N The action of streptococcal amylase in relation to the synthesis of an amylosaccharide by amylolytic strains of Streptococcus pyogenes. J. Gen. Microbiol. 13: DAIN, J. A., A. L. NEAL, AND H. W. SEELEY The effect of carbon dioxide on polysaccharide production by Streptococcus bovis. J. Bacteriol. 72: DOETSCH, R. N., R. Q. ROBINSON, R. E. BROWN, AND J. C. SHAW Catabolic reactions of mixed suspensions of bovine rumen bacteria. J. Dairy Sci. 36: HoBSON, P. N., AND M. MACPHERSON Amylases of Clostridium butyricum and a streptococcus isolated from the rumen of a sheep. Biochem. J. 62: HoBSON, P. N., AND S. 0. MANN Some factors affecting the formation of iodophilic polysaccharides in group D streptococci from the rumen. J. Gen. Microbiol. 13: KNEEN, E., AND L. D. BRECKFORD Quality and quantity of amylase production by various bacterial isolates. Arch. Biochem. 10: MACPHERSON, M Isolation and identification of amylolytic streptococci from the rumen of the sheep. J. Pathol. Bacteriol. 66: MANN, S. O., AND A. E. OXFORD Relationships beteen viable saccharolytic bacteria in the rumen and abomasum of the young calf and kid. J. Gen. Microbiol. 12: MONOD, J., AND A. M. TORRIANI De l'amylomaltase d'escherichia coli. Ann. inst. Pasteur 78: NEUFELD, E. F., AND W. Z. HASSID Hydrolysis of amylose by,b-amylase and a-amylase. Arch. Biochem. Biophys. 59: NIVEN, C. F., JR., M. R. WASHBURN, AND J. C WHITE Nutrition of Streptococcus bovis. J. Bacteriol. 55: OXFORD, A. E The nutritional requirements of rumen strains of Streptococcus bovis considered in relation to dextran synthesis from sucrose. J. Gen. Microbiol. 19: ROBINSON, R. Q., R. N. DOETSCH, F. M. SIROTNAK, AND J. C. SHAW Production of lactic acid and an iodine staining substance by bovine rumen bacteria. J. Dairy Sci. 38: SCHWIMMER, S., AND A. K. BALLS Isolation and properties of crystalline alpha-amylase from germinated barley. J. Biol. Chem. 179: SEELEY, H. W., AND J. A. DAIN Starch hydrolyzing streptococci. J. Bacteriol. 79: SHERMAN, J. M., AND P. STARK Streptococci hich gro at high temperatures. J. Bacteriol. 22: SHERMAN, J. M The streptococci. Bacteriol. Rev. 1:3-97. SMITH, J. A. B., AND F. BAKER The utilization of urea in the bovine rumen. 4. The isolation of the synthesized material and the correlation beteen protein synthesis and microbial activities. Biochem. J. 38: SOMOGYI, M Estimation of reducing sugars. J. Biol. Chem. 160: WOLIN, M. J., G. B. MANNING, AND W. 0. NELSON Ammonium salts as a sole source of nitrogen for the groth of Streptococcus bovis. J. Bacteriol. 78:147. Donloaded from on September 12, 2018 by guest