Altered Metabolism in a Streptococcus lactis C2 Mutant Deficient in Lactic Dehydrogenase1

Altered Metabolism in a Streptococcus ladis C2 Mutant Deficient in l.adic Dehydrogenas¢ L. L. McKAY and K. A. BALDWIN Department of Food Science and Nutrition University of Minnesota, St. Paul 55101

ognized for this potential. However, Krishnaswamy and Babel (13) found that certain cultures of S. lactis and S. cremoris produced aroma compounds. Fryer (9) cited work showing that 11.l% of the milk citrate was broken down by S. cremoris HP and that S. lactis metabolized l~C-labeled citrate. Strains of S. lactis and S. cremoris producing excessive amounts of CO2 have also been observed (17). The reason for the spontaneous appearance of these altered strains is not known. S. lactis C2, the organism reported an here, is homofermentative, and most of the pyruvate formed from carbohydrate metabolism must be reduced to lactic acid by lactic debydrogenase to reoxidize the reduced nicotinamide adenine dinucleotide (NAD) formed during glycolysis. Pyruvate can be a precursor of several end-products produced by starter organisms. Anders and Jago (1) pointed out that inhibition of lactic dehydrogenase in S. cremoris 13 by certain fatty acids could alter the end-products and thereby influence the flavor of cultured dairy products. Our results describe the isolation and metabolic characteristics of a mutant of S. lactis C2 which is defective in lactic dehydrogenase. This defect results in the production of excessive amounts of CO2 and acetoin.

Abstract

A spontaneous mutant of Streptococcus lactis C2 which formed abnormally large colonies on agar medium was isolated. The mutant grew as rapidly in broth or milk as the parent culture but was slower in acid production. When grown in complex broth at 32 C, the parent culture lowered the pH to 4.8 in 8 h whereas the mutant required 15 h to produce pH 5.0. Mutant cells consumed 6 times as much oxygen as S. lactis C2 and produced carbon dioxide derived primarily from carbons 3 and 4 of glucose. The mutant was defective in lactic dehydrogenase possessing only .1 units as compared to 138 units for S. lactis C2. This enzymatic defect caused the mutant to produce about 1,000 /,g/ ml of Westerfeld positive material when grown in broth. S. lactis C2 produced only trace amounts. Alth,'mgh acetoin was a major metabolic end-product in the mutant, diacetyl was also detected. The results indicate a type of mutation which could be responsible for the spontaneous occurrence of aroma and carbon dioxide producing strains of S. lactis in dairy starter cultures.

Materials and Methods Introduction

Occasionally from a pure culture of Streptococcus lactis or S. cremoris a strain is isolated which produces a flavor and aroma suggesting production of diaeetyl, volatile acids, and carben dioxide (CO2). S, diacetilactis can form these compounds from citrate (6), but strains of S. lactis or S. cremoris are not usually recReceived August 9, 1973. ' Scientifie Journal Series Paper No. 8415, Minnesota Agricultural Experiment Station, St. Paul, Minnesota 55101. Presented in part at the 68th Annual Meeting of the American Dairy Science Association, Washington State University, Pullman, Washington, June 24-27, 1973.

Cultures'. Streptococcus lactis C2 and S. diacetilactis 18-16 used in this study are maintained in our stock culture collection. Their maintenance and original source were described previously (16). The mutant, S. lactis KB, was isolated as a spontaneous variant of S. lactis C2. A lytic bacteriophage against S. lactis C2 was obtained from W. E. Sandine, Department of Microbiology, Oregon State University, Corvallis. Growth experiments. The organisms were serially transferred at least three times in sterile (121 C, 12 rain) 11% (w/v) reconstituted nonfat milk (Matrix Mother Culture, Medium, Galloway-West, Fond du Lac, Wisconsin) or in broth before inoculating into ex-

181

182

MCKAY AND BALDWIN

perimental flasks. For growth studies, the or- Lowry et al. (15). The supernatants were ganisms were grown at 32 C, serially diluted dialyzed 16 h against .1M sodium phosphate at hourly intervals, and plated on lactic agar buffer (pH 7.0) at 4 C. NAD dependent lactic (8). Following ineubation at 32 C for 48 h, dehydrogenase activity was measured from the all visible colonies were counted. Also, at in- change in absorbance at 340 nm of a reaction tervals, aliquots of the culture were removed mixture which contained 250 /xmoles trifrom the flasks for pH measurements. In simi- ethanolamine-HC1 (pH 6.0), 20 /~moles solar experiments, samples were removed for dium pyruvate, .4 /~moles reduced NAD, quantitative determination of lactic acid and 3.0 /zmoles fructose- 1,6-diphosphate, and aeetoin plus diacetyl production by the meth- cell extract in a total volmne of 3.0 ml (2). ods of Ling /14) and Westerfeld (18), re- The initial reaction rates were measured by a spectively. The effect of growth medium on Beckman Aeta III Recording Speetrophotomeacetoiu plus diacetyl production in S. lactis ter. Identification of acetoin and diacetyl. Ten C2, S. lactis KB, and S. diacetilactis 18-16, was determined by growing the organisms in milliliters of spent medium from a 16-h lac11% nonfat milk, "glucose broth (8), lactose- tose-citrate broth culture of the organisms were citrate broth (11), or lactose-citrate broth extracted 10 times with dichloromethane. The extracts were concentrated to 1.0 ml, and without citrate, at 32 C for 16 h. Respiration studies. Cells were grown in samples were then analyzed by gas-liquid lactic broth at 32 C for 7 h and were har- chromatography (GLC) with a Hewiettvested, washed, and resuspended in sufficient Packard gas chromatograph (Model 7620A) .05M sodium phosphate buffer (pH 6.6) with equipped with dual flame ionization detectors. .025M MgCI2 to provide an optical density of The column (stainless steel, 1.83 M X .32 7.0 at 650 nm. Dry weights were determined cm OD) was packed with 10% carbowax by drying 1.0-ml samples of these suspensions 20 m on 80/100 mesh chromosorb P and opat 85 C. Oxygen uptake studies were in a erated with a carrier gas (Helium) at a flow Warburg respirometer at 32 C. The flasks con- rate of 35 ml/min. The oven temperature was tained 50 ~moles sodium phosphate buffer programmed from 65 to 130 C at 8 C/min, (pH 7.0), 25/.trnoles MgCI2, 15 p.moles glucose, following a 5-min postinjection hold, and the and approximately 5.0 mg dry weight of cells in injection port was maintained at 250 C. The a total volume of 3.0 ml. The center well of the control consisted of adding acetoin and Warburg flasks contained .1 ml of 20% KOH. diaeetyl (500 and 100 /xg/ml final concentraIn experiments with labeled glucose, the r4CO2 tions) to 9 ml lactose-citrate broth, extracting was trapped on a fluted piece (2 by 2 cm) and examining by CLC as described above. of Whatman No. 1 filter paper saturated with Mass spectral data were from a LKB 9000 10% KOH (4). Sulfuric acid (.2 ml of 10 N) mass spectrometer employing the same column was tipped into the reaction mixture from the and operating parameters described for the side-arm at the end of 1 h to release all of the GLC analyses. CO.~, and the flasks were incubated 1 h longer. The '4CO._, was then counted in a liquid Results and Discussion ~cintillation spectrophotometer by dropping During the isolation of spontaneous metabolthe filter paper into a vial c~mtaining the ic variants of S. lactis C2, a eertain colony scintillation fluor of 4.0 g 2,5-diphenyloxazole developed outgrowths after 12 days incuba(PPO) and .05 g 1,4-bis-2-(5-phenyloxazolyl tion on lactose agar plates at 25 C. This colony was picked into broth, grown, and then benzene) per liter of toluene. Lactic dehydrogenase estimation. The or- plated on lactose agar. After incubation, the ganisms were grown in 1 liter of .5% glucose plates showed a combination of abnormally broth for 10 h at 32 C. The cells were har- large and normal size colonies. The large vested, washed twice with .85% NaCI, and colonies were purified and treated with H202 resuspended in 4.0 ml .083M triethanolamine- to insure the cells were catalase negative. The HC1 buffer at pH 7.0. Cell-free extracts were cells were lysed by S. lactis C2 phage, thereby prepared by extruding the cells through an ruling out the possibility of contamination. For E~ton Press (7) at a constant pressure of the purpose of identification, this spontaneous 703.1 x 104 kg/m -°. The frozen mixture was mutant was designated S. lactis KB. Sizes of removed from the press, .thawed, and cen- colonies formed by S. lactis C2 and the mutant trifuged at 10,000 X g for 10 min to remove S. lactis KB are compared in Fig. 1. After ~cellular debris. Protein content of the super- 48 h incubation at 32 C, S. lactis C2 colonies natant fluid was measured by the method of were about 1 mm in diameter whereas the .~OURNKL OF ~)AIRY SEI~NCE VOL. 57. NO. 2

S. L A C T I S M U T A N T

183

15

60 t¢3 ..a

e

45

E

~3o ..J

Fxc. 1. Appearance of large colonies (S. lactg~ KB) among normal size colonies (S. lactis C2) on a lactic agar plate incubated 48 h at 32 C. nmtant cdlonies were about 3 mm in diameter. The larger mass of cells produced by the mutant suggested that it may reach a higher population density than S. lactis C2 in milk or broth and would, therefore, be useful in mass culturing of lactic streptococci. The two organisms grown in milk or lactic broth at 32 C exhibited similar growth responses, and the mutant did not achieve a significantly higher population (Fig. 2). Even though the mutant grew as rapidly in broth or milk as the parent eulture, it was slower in acid production. When grown in lactic broth the parent culture lowered the pH to 4.8 in 8 h whereas the mutant required 15 h to lower the p t I to 5.0. During these growth experiments S. lactis KB was producing gas. This gas-producing trait of the mutant merited further investigation since excessive gas production is seldom observed in normal lactic starter cultures.

9 .o ! 8.0

<

/Y

0

i

20

TIME

,t .ol

6.0

Radioactivity released as 14CO_(cpm/mg cell dry wt) S. lactis C2 S. lactis KB

Glucose-U-14C Glucose-l-l*C Glucose-3,4-1~C

4.0

TIME

0 5 Ir~ HOURS

10

904 1,115 1,527

~

t 1,918 1,623 41,019

pH

5.0 15

100

Studies on respiration were initiated, and the oxygen taken up by suspensions of whole cells was measured. S. lactis KB consumed about six times as much oxygen as S. Iactis C2 (Fig. 3). After 60 min of incubation, KB had taken up 54/~1 02 per mg cell dry weight, as compared to only 9 p,l for C2. The COo released from Carbohydrate metabolism was measured by incubating the cells with carbon-14 labeled glucose and trapping any 14CO2 released. The release of laCO2 from glucose labeled uniformly, in C-l, or in carbons 3, 4 is in Table 1. With uniformly labeled (U14C) glucose, S. lactis KB produced more COo than S. lactis C2. The mutant bad released about 12,000 cpm CO2 as compared to only 900 cpm CO~ by C2. The enhanced

Labeled snbstrate

7.0

}0

80

IN M I N U T E S

FJc. 3. Time course of oxygen eonsmned by S. laetis C2 (C)) and S. lactis KB ( • ) as measured in a Warburg respirometer.

o6.0

5

60

TABLE 1. Release of "CO., by S. lactis C2 and S. lactis KB from glucose which was labeled uniformlv, in C-l, and in carbons 3,4. 3

o z oS.G

3.C

40

15

Fro. 2. Typical growth curves and pH changes when S. lactis C2 ( O ) or S. /act/s KB ( • ) were grown in milk (A) or lactic broth (B) at 32 C.

Reaction mixture contained 1.0 ml cell suspension (approximately 5.0 mg dry weight), 1.0 ml .05 M sodium phosphate buffer (pH 7.0) containing .09,5 M MgCI,,, and 1.0 ml labeled glucose (15 #moles glucose containing .013 /~Ci, x4C per ~mole of glucose). JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 2

184

MCKAY

AND

release of COz and its slow acid production initially suggested that glycolysis was inhibited in S. lactis KB and that the mutant was forced to utilize glucose primarily via the hexose monophosphate shunt. However, the amount of r4COz released from glueose-l-l~C was similar in both organisms (Table 1). If glucose was being utilized via the hexose monophosphate shunt, CO2 production from C-1 of glucose would have increased. The release of CO2 from glucose labeled in carbons 3, 4 was examined. The results (Table 1) indicate that the mutant released 41,000 epm COz compared to only 1,500 cpm COz for S. lactis C2. This indicated the mutant may be defective in lactic hydrogenase (LDH) and, instead of converting pyruvate primarily to lactic acid, was converting it to CO,_, and other products. The LDH activity of S. lactis C2 and S. lactis KB is shown in Table 2. Enzyme activity was defined as LDH units per mg protein, and 1 unit is defined as that amount of enzyme causing a change in the absorbancy of .1 units/min at 340 nm. S. lactis C2 possessed 138 units of LDH activity as compared to only .1 units in KB, with consideration of the endogenous rate in this organism. In the absence of pyruvate, no activity was observed in S. lactis C2, but the mutant possessed some endogenous activity. This was perhaps due to increased NADHz oxidase activity and would be consistent with the increased oxygen consumption by this organism (Fig. 2). Since lactic dehydrogenase is defective, this reaction would serve to reoxldize the NADH, formed during glycolysis. Bruhn and Collins (3) have reported NADH2 oxidase in

TABLE 2. Relative lactic dehvdrogenase (LDH) activity o£ S. lacti~ C2 and S. lactis KB. Reaction system"

Lactic dehydrogenase activityb S. lactis C2 S. lactis KB

Complete Complete minus pyruvate Complete minus fructose-l,6-diphosphate

138 0

.7 .6

1.5

.6

a The complete reaction mixture consisted of 250 #moles triethanolamine-HCl (pH 6.0), 20 tzmoles sodium pyruvate, .4 umole NADH, 3.0 t~moles fructose-l,6-diphosphate, and cell extract in a total volume of 3.0 ml. b Enzyme activity was defined as LDH units/mg protein. JOURNAL OF DAIRY SCIENCE VOL.

57, N O .

2

BALDWIN

LACID AC~TI C CFU LOG I ML q O~A 08

ACETOIN ug t ~L

675 600

525 450 375

0020468"0 06"0l0 f~

300 225 150 75

00

0

b

,

8

m

12 16 III

4 8 12 16 20 24 rIMEIN HOURS

20 24

0

Fie. 4. Comparison of growth (Q)), lactic acid ( • ) , and acetoin plus diacetyl (R) production by S. lactis C2 (A) and S. lactis KB (B) when propagated in milk at 32 C.

S. diacetilactis. As shown by Anders et al. (2), fructose-I, 6-diphosphate markedly activated the LDH's of lactic streptococci. The effect of this enzymatic defect on endproducts produced by S. lactis KB as compared to S. lactis C2 is in Fig. 4. These organisms were grown in milk and the viable count, as well as lactic acid and diacetyl plus acetoin production, was measured. S. lactis C2 rapidly produced lactic acid achieving .68% lactic acid in 24 h. Only trace amounts of Westerfeld-positive material were detected. On the other hand, S. lactis KB produced only .12% lactic acid in 24 h but was markedly higher than S. lactis C2 in the production of Westerfeld-positive material. Over 650 /xg per ml of acetoin plus diacetyl was produced in 21 h. The effect of several growth media on acetoin plus diacetyl production by S. lactis C2, S. lactis KB, and S. diacetilactis 18-16, a known producer of acetoin plus diacetyl, is shown in Table 3. The total viable count and pH were also measured. All determinations were after 16 h of incubation. S. lactis C2 produced only trace amounts of acetoin plus diacetyl in all media tested. When grown in milk, S. lactis KB produced 450 t~g/ml and S. diacetilactis 18-16 225 /~g/ml. In glucose broth, the mutant produced 866 /~g/ml whereas only trace amounts were produced by S. diacetilactis 18-16. In lactose-citrate broth, S. lactis KB produced over 1,000 t~g/ml and S. diacetilactis 18-16 produced 602. On this same medium, but without citrate, the mutant produced 796 /~g/ml and S. diacetilactis 18-16 produced only trace amounts. Without citrate, S. diacetilactis 18-16 only produced

S. L A C T I S M U T A N T

185

TABLE 3. Influence of growth medium on total cell population, pH, and acetoin plus diacetyl production in S. lactia C2, S. /act/s KB, and S. diacetilactis 18-16. S. lactis C2

Media Milk Glucose broth Lactosecitrate broth Lactosecitrate broth without citrate

S. lactis KB

S. diacetilactis 18-16

Diacetyl+ Diacetyl+ acetoin TPC pHof acetoin (~g/ml) × 10~ medium (~g/ml)

TPC ~ × 10s

pHof medium

TPC pHof × l0 s medium

19.5

4.5

.1

19.5

5.2

450

8.1

5.6

3.3

4.4

.5

7.9

4.8

866

3.4

4.4

1.2

4.7

1.6

12.0

4.9

1,058

6.2

4.7

3.0

4.6

1.7

7.5

4.8

796

2.8

4.6

Diacetyl+ acetoin (~g/ml) 225 1.5

602

22

" TPC = total plate count. trace amounts of acetoin plus diacetyl whereas S. lactis KB produced copious amounts. The latter organism was not dependent upon citrate for production of Westerfeld-positive material, but, as documented by Collins (6), S. diacetilactis needs citrate to serve as a source of excess pyruvate to produce acetoin plus diacetyl. Chuang and Collins (5) reported that S. lactis C2 grown in lactose-citrate medium produced acetoin maximally at the end of logarithmic growth. The amount produced was about 15 /~g/ml in comparison to over 1,000 /~g/ml of Westerfeld-positive material produced by S. lactis KB in the same medium. To determine whether diacetyl, acetoin, or both, were being produced by the mutant, the

A

c]

e

! ~¢,- ^ct~iN

spent medium of a 16-h culture was treated with diehloramethane to extract these compounds. Aliquots were examined by GLC (Fig. 5). The Westerfeld-positive material produced by S. lactis KB was primarily acetoin, but a trace of diaeetyl was detected, as confirmed by mass spectrometry. Gunsalus (10) reported that the pathway of acetoin formation in lactic acid bacteria required a pyruvate concentration which was higher than that required by other pathways utilizing this substrate. From this, one or more of the enzymes involved has a high Miehaelis constant. The results are consistent with this hypothesis, since in S. lactis KB the pyruvate in the cell would reach a concentration at which enzymes forming acetoin and diacetyl become active. The concentration of pyruvate builds up in S. lactis KB because the organism is defective in lactic dehydrogenase. As reported by Harvey and Collins (12) for S. diacetilactis, this removal of excess pyruvate with formation of acetoin and diacetyl may serve as a detoxification mechanism for the cell.

I

5.1

8.I

.I ,~ETENTION

5.1 TLME

9.i

I .I

5.1

8.1

I~, MINUTES

FIG, 5. Gas-liquid chromatogram of concentrated dichloromethane extracts from lactose-citrate broth cultures of S. lazti~ KB and S. diacetilactis 18-16 showing the presence of acetoin and diacetyl. (A represents the retention times of the diacetyl and acetoin standards; B, inoculated with S. lactis KB; C, inoculated with S. diacetilactis 18-16. )

The data indicate a type of metabolic defect or mutation which influences the metabolism of the starter culture and, thereby, the nature of the end-products. In this case, S. lactis C2 has become defective in lactic dehydrogenase, resulting in the production of large amounts of CO2 and acetoin. This may be a type of mutation which is responsible for the spontaneous occurrence of aroma and CO2 producing strains of S. lactis or S. cremoris in dairy starter cultures. JOURNAL OF DAIRY SCIENCE VOL, 57, NO. 2

186

MCKAY AND BALDWIN

Acknowledgments The authors acknowledge Dr. G. A. Reineccius for assisting with GLC analyses and T. Krick, Department of Biochemistry, for performing the mass spectrometry. References (1) Anders, R. F., and G. R. Jago. 1970. The effect of fatty acids on the metabolism of pyruvate in lactic acid streptococci. J. Dairy Res. 37:445. (2) Anders, R. F., H. A. Jonas, and G. R. Jago. 1970. A survey of the lactate dehydrogenase activities in Group N streptococci. Australian J. Dairy Teehno]. 25:73. (3) Bruhn, J. C., and E. B. Collins. 1970. Reduced nicotinamide adenine dinucleotide oxidase of Streptococc~ diacetilact~. J. Dairy Sci. 53:857. (4) Buhlen, D. R. 1962. A simple scintillation counting technique for assaying 1'CO.. in a Warburg flask. Anal. Biochem. 4:413. (5) Chuang, L. F., and E. B. Collins. 1968. Biosynthesis of diacetyl in bacteria and yeast. J. Bacteriol. 95:2083. (6) Collins, E. B. 1972. Biosynthesis of flavor compounds by microorganisms. J. Dairy Sci. 55:1022. (7) Eaton, N. R. 1962. New press for disruption of microorganisms, j. Bacteriol. 83: 1359. (8) Elliker, P. R., A. W. Anderson, and G. H. Hannesson. 1956. An agar culture medium for lactic acid streptococci and lactobacilli. J. Dairy Sci. 39:1611.

JOURNAL OF DAIRY SCIENCE VOL. 57, N o .

2

(9) Fryer, T. F. 1969. Microflora of cheddar cheese and its influence on cheese flavor. Dairy Sci. Abstr. 31:471. (10) Gunsalus, I. C. 1958. Energy metabolism of lactic acid bacteria. Pages 444-449 ~n O. Hoffmann-Ostenhof, Proe. 4th Int. Congr. Biochem. Pergamon Press, New York. (11) Harvey, R. J., and E. B. Collins. 1961. Role of citritase in aceto~n formation by Streptococcus diacetilactis and Leuconostoc citrovorum. J. Bacteriol. 82:954. (12) Harvey, R. ]., and E. B. Collins. 1963. Role of citrate and acetoin in the metabolism of Streptococcus diacetilactis. J. Bacteriol. 86:1301. (13) Kristmaswamy, M. A., and F. J. Babe]. 1951. Biacetyl production by cultures of lactic acid-producing streptococci, j. Dai13~ Sci. 34: 374. (14) Ling, E. R. 1951. The determination of lactic acid in milk. J. Sci. Food Agr. 2:279. (15) Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. ]93:265. (16) McKay, L. L., K. A. Baldwin, and E. A. Zottola. 1972. Loss of lactose metabolism in lactic streptococci. Appl. Microbiol. 23: 1090. (17) Sandine, W. E., P. R. Elliker, and A. W. Anderson. 1957. A simple apparatus for measurement of gas production and activity of lactic starter cultures. Milk Prod. J. 48:12. (18) Westeffeld, W. W. 1945. A colorimetric determination of blood acetoin. I. Biol. Chem. 161:495.