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Batch and chemostat cultures of Streptococcus cremoris HP and Streptococcus lactis 829 were examined for lactose-negative (lac−) mutants on indicator agar. In batch cultures, S. cremoris HP gave less than 1% of the total count as lac− colonies while S. lactis 829 consistently contained about 15% of the total as lac− colonies. In chemostat cultures of S. cremoris HP in 2% skim milk containing casamino acids and yeast extract (0.1% each), the percentage of lac− colonies increased markedly when the temperature of growth was 18 °C but not when the temperature of growth was 25 °C. The percentage of lac− colonies in chemostat cultures in the skim milk medium at 25 °C was about the same as that in batch cultures. On the other hand, when chemostat cultures of S. lactis 829 in the skim milk medium were grown at several temperatures between 18 and 33 °C, the percentage of lac− colonies was markedly lower than that found in batch cultures of this organism. Cultivation of S. cremoris HP in chemostats with yeast extract – lactose broth at low temperatures (14–18 °C resulted in cultures that gave plate counts on lactose agar, which were as much as 50% lower than counts on glucose agar but did not result in the selection of lac− mutants. Cultivation of S. lactis 829 in chemostats with yeast extract – glucose broth at low temperature (18 °C resulted in a selection of cells giving lac− colonies and atypical (small) lac+ colonies. The results show that cultivation of S. cremoris HP and S. lactis 829 in chemostats sometimes gave rise to altered populations. Conditions causing a change in one organism did not necessarily cause a similar change in the other. The results indicate that the successful propagation of lactic streptococci in chemostats for use as starter cultures in the dairy industry will require the careful establishment of optimum conditions for every strain so as to minimize the possible selection of undesirable populations.

An electrochemical potential difference for hydrogen ions ( a protonmotive force) was artifically imposed across the membrane of the anaerobic bacterium Streptococcus lactis. When cells were exposed to the ionophore, valinomycin, the electrical gradient was established by a potassium diffusion potential. A chemical gradient of protons was established by manipulating the transmembrane pH gradient. When the protonmotive force attained a value of 215 mV or greater, net ATP synthesis was catalyzed by the membrane-bound Ca++, Mg++ -stimulated ATPase. This was true whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Under these conditions, ATP synthesis could be blocked by the ATPase inhibitor, dicyclohexylcarbodiimide, or by ionophores which rendered the membrane specifically permeable to protons. These observations provide strong evidence in support of the chemiosmotic hypothesis, which states that the membrane-bound ATPase couples the inward movement of protons to the synthesis of ATP.

SummaryYeast extract was fractionated on Sephadex G-25 into 7 fractions. The fraction most stimulatory to the growth ofStreptococcus lactisC10 contained over 70% of the amino N present in yeast extract and consisted of a wide variety of free amino acids and a small amount of peptide material. Examination of possible replacement factors for this fraction revealed that the amino-acid material present was largely responsible for the stimulation ofStr. lactisC10. Purine and pyrimidine bases and inorganic constituents also contributed to the stimulation. In addition, yeast extract contained a component which decomposed H2O2, a metabolite which accumulates in the growth medium under aerobic conditions and inhibits growth. The nature of the stimulation was studied by isolating slow and fast acid-producing colonies ofStr. lactisC10. It appeared that yeast extract and other amino-acid supplements prevented an observed inhibition of the growth of the slow variants below pH 6·0, apparently by satisfying a nutritional deficiency caused by a drop in pH.

Incorporation of 1.9% β-disodium glycerophosphate (GP) into a complex medium resulted in improved growth by lactic streptococci at 30 C. The medium, called M17, contained: Phytone peptone, 5.0 g; polypeptone, 5.0 g; yeast extract, 2.5 g; beef extract, 5.0 g; lactose, 5.0 g; ascorbic acid, 0.5 g; GP, 19.0 g; 1.0 M MgSO 4 ·7H 2 O, 1.0 ml; and glass-distilled water, 1,000 ml. Based on absorbance readings and total counts, all strains of Streptococcus cremoris, S. diacetilactis , and S. lactis grew better in M17 medium than in a similar medium lacking GP or in lactic broth. Enhanced growth was probably due to the increased buffering capacity of the medium, since pH values below 5.70 were not reached after 24 h of growth at 30 C by S. lactis or S. cremoris strains. The medium also proved useful for isolation of bacterial mutants lacking the ability to ferment lactose; such mutants formed minute colonies on M17 agar plates, whereas wild-type cells formed colonies 3 to 4 mm in diameter. Incorporation of sterile GP into skim milk at 1.9% final concentration resulted in enhanced acid-producing activity by lactic streptococci when cells were inoculated from GP milk into skim milk not containing GP. M17 medium also proved superior to other media in demonstrating and distinguishing between lactic streptococcal bacteriophages. Plaques larger than 6 mm in diameter developed with some phage-host combinations, and turbid plaques, indicative of lysogeny, were also easily demonstrated for some systems.

The inhibition of growth of Staphylococcus aureus by lactic streptococci in associative cultures in milk was not due to hydrogen peroxide produced by the streptococci. Dialyzed whey from the milk culture of lactic streptococci was more inhibitory than dialyzed whey from milk acidified with lactic acid, indicating that material other than lactate was also involved. Analyses of cation and anion exchange fractions from the dialyzed whey showed that only the neutral fraction was inhibitory.

When cells of Streptococcus lactis or Escherichia coli were suspended in a potassium-free medium, a membrane potential (negative inside) could be artificially generated by the addition of the potassium ionophore, valinomycin. In response to this inward directed protonmotive force, ATP synthesis catalyzed by the membrane-bound ATPase (EC 3.6.1.3) was observed. The formation of ATP was not found in S. lactis that had been treated with the ATPase inhibitor, N,N′ -dicyclohexylcarbodiimide, nor was it observed in a mutant of E. coli lacking the ATPase. Inhibition of ATP synthesis in S. lactis was also observed when the membrane potential was reduced by the presence of external potassium, or when cells were first incubated with the proton conductor, carbonylcyanidefluoromethoxyphenylhydrazone. These results are in agreement with predictions made by the chemiosmotic hypothesis of Mitchell.

Partial lactose-fermenting revertants from lactose-negative ( lac − ) mutants of Streptococcus lactis C2 appeared on a lawn of lac − cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac + S. lactis C2, the revertants were defective in the accumulation of [ 14 C]thiomethyl-β- d -galactoside, indicating that they were devoid of a transport system. Hydrolysis of o -nitrophenyl-β- d -galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-β- d -galactosidase (P-β-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac + S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac + S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac − , respectively. This suggested that the lac − and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-β-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.

SUMMARY: In some nisin-producing strains of Streptococcus lactis, clones which were permanently incapable of producing nisin (Nis-) appeared spontaneously with a frequency of 0.03 to 0.82%. Treatment with proflavin (Pro), ethidium bromide (EB) or at a temperature of 40 °C increased the number of such clones. In one strain 78% of cells became Nis- after treatment with Pro. Treatment with nitrosoguanidine did not reverse the effect of Pro or EB. There was no evidence that Pro or EB selected spontaneously-occurring Nis- clones. These observations suggest that the gene(s) responsible for nisin production in some S. lactis strains may be located on a plasmid, but further investigation is required to confirm this.

Variants of Streptococcus lactis that produce lactic acid slowly in milk were isolated by inducing plasmid loss in the wild type at 39 to 40 C. Such strains had lost most of their surface-bound proteinase activity and were designated prt - . The specific proteinase activities of S. lactis C10 prt + whole cells and solubilized cell walls were 7 and 18 times, respectively, those of the prt - strain, but spheroplast lysates of prt + and prt - strains contained similar proteinase activity. S. lactis H1 showed a similar relative distribution of activity between prt + and prt - cellular fractions, although the overall level was lower. The limited growth in milk, characteristic of prt - strains, can be explained in terms of their low surface-bound proteinase activity.

Ultraviolet (UV)-induced phage lysates, from lactose-positive ( lac + ) Streptococcus lactis C2, transduced lactose fermenting ability to lac − recipient cells of this organism. Although the phage titer could not be determined due to the absence of an appropriate indicator strain, the number of transductants was proportional to the amount of phage lysate added. Treatment of the lysate with deoxyribonuclease had no effect on this conversion, indicating the observed genetic change was not mediated by free deoxyribonucleic acid. When the lac + transductants were isolated and exposed to UV irradiation, lysates with higher transducing ability were obtained. The transducing ability of this lysate was about 100-fold higher than that observed in the original lysates. The lac + transductants were unstable since lac − segregants occurred at high frequency. The phage lysate from S. lactis C2 also transduced maltose and mannose metabolism to the respective negative recipient cells. The results demonstrate the transduction of carbohydrate markers by a streptococcal phage and establish a genetic transfer system in group N streptococci.

SummaryA versatile continuously-stirred fermentation apparatus, suitable for batch and continuous culture studies, has been constructed from glassware normally available in the laboratory. With the aid of normal workshop facilities an effective stainless steel stirrer gland was constructed. This apparatus has been used successfully for batch and continuous culture studies on the growth of lactic streptococci in skim-milk medium.

SummaryInvestigations on the growth characteristics ofStreptococcus lactisML8in skim-milk medium have indicated that agitation of the medium may decrease the growth rate of the organism by decreasing the availability of CO2(CO2effect) and of soluble nitrogen (soluble nitrogen effect). The former effect was observed only when the CO2concentration was depleted by heat sterilization; the latter occurred when a certain threshold level of agitation was exceeded. Results suggested that the soluble nitrogen affect was caused by an alteration in the rate of diffusion of substances formed in the immediate vicinity of the bacterial cell by the proteolytic enzyme system.

Williamson , W. T. (North Carolina State of the University of North Carolina, Raleigh), S. B. Tove, and M. L. Speck . Extracellular proteinase of Streptococcus lactis . J. Bacteriol. 87: 49–53. 1964.— Streptococcus lactis was shown to produce an extracellular proteolytic enzyme(s). A 120-fold purification of the proteinase was obtained from the cell-free culture medium by ammonium sulfate fractionation, calcium phosphate gel treatment, and chromatography on diethylaminoethyl cellulose. Only 32% of the activity was destroyed by heating at 98 C for 1 hr. The requirement of free sulfhydryl groups for enzyme activity was indicated by inhibition with p -chloromercuric benzoate. Diisopropyl fluorophosphate did not inhibit the proteinase.

Streptococcus lactis (ATCC 7962) accumulated thiomethyl-β-galactoside (TMG) and other galactosides against concentration gradients when the cells were supplied with a metabolizable substrate, such as glucose. The accumulated TMG was free and not phosphorylated. In the absence of glucose, TMG rapidly entered the cell to a concentration equal to that of the medium. Agents that uncouple oxidative phosphorylation abolished active transport but not the carrier-facilitated entry of TMG. Evidence that the transport carriers were functional in the absence of glucose or in the presence of uncoupling agents included the demonstration of counterflow, which depends on competitive inhibition for the carrier for exit.

MacLeod, Patricia (University of Connecticut, Storrs), R. G. Jensen, G. W. Gander, and J. Sampugna . Quantity and fatty acid composition of lipid extracted from cells of Streptococcus lactis . J. Bacteriol. 83: 806–810. 1962.—A method for the extraction of lipid from bacterial cells is described. Lysozyme-treated cells of Streptococcus lactis yielded 5% of their dry weight as lipid, whereas cells not treated with lysozyme yielded only 3% lipid. More than two-thirds of the bacterial lipid extracted was tentatively classified as phospholipid, based on the elution behavior of this fraction on silicic acid columns when chloroform and methanol were the eluting solvents. The following five major fatty acids were present in all lipid fractions isolated: myristic, palmitic, palmitoleic, lactobacillic, and an 18-carbon acid possessing one double bond, which may actually be oleic acid or a mixture of oleic and one or more of its isomers. These acids comprised more than 85% of the acids found, as indicated by gas-liquid chromatography of their methyl esters. Since lactobacillic acid accounted for about 20% of the fatty acids extracted from cells of S. lactis , this organism may prove to be a ready source of this comparatively rare acid.

The effects of p H, temperature, proportion of Staphylococcus aureus in the inoculum, various strains of effector organism, and various strains of S. aureus were examined for their influence on interactions between staphylococci and effector organisms in associative culture. In general, small changes in p H had little effect upon either growth of S. aureus or production of enterotoxin in associative culture. Inhibition of growth of S. aureus caused by effector organisms was much greater at 25 than at 30 C. Proportion of S. aureus in the inoculum greatly affected both growth of the staphylococci and production of enterotoxin. Only slight differences were found between strains of either effector organism or S. aureus which affected the interactions in associative culture.

Proteins in sonic extracts of Streptococcus lactis were separated by starch-gel electrophoresis at high voltage. Each slab was sliced longitudinally, and half was stained for peptidases in a mixture containing a peptide, L-amino acid oxidase (snake venom), peroxidase, and o -dianisdine; the other half was stained in amido black for protein. In addition to sonic treatment, trypsin also released enzyme from acetone-treated cells. Glycyl-L-phenylalanine, L-phenylalanyl-glycine, L-alanyl-L-phenylalanine, and L-phenylalanyl-L-alanine served as substrates in characterizing the enzymes. Five different fractions of various specificities appeared in the gels. Broad-range substrate specificities were found for sonic extracts of S. lactis, S. cremoris, S. durans , and Lactobacillus acidophilus .

1. 1. The less polar phosphoglucolipid from Streptococcus faecalis was isolated. It contained D -glucose, glycerol, fatty acid ester and phosphorus in a molar ratio of approx. 2:2:4:1. 2. 2. The structure was shown to be 3(1)-O-[6'-(1,2-diacyl-sn-glycero-3-phosphoryl) -2'-O-(α- D -glucopyranosyl)-α- D -glucopyranosyl]-1(3),2-diacylglycerol. 3. 3. Structural analysis of the deacylated core was based on periodate oxidation, Smith degradation, alkaline hydrolysis and enzymatic degradation. The location of the acyl groups was achieved by degradation of the phosphoglucolipid with 60% HF (w/v) resulting in the formation of diglycerides and diglucosyl diglyceride. 4. 4. Isolation of these breakdown products, cleavage of the diglucosyl diglyceride with a position-specific lipase and treatment of the intact lipid with phospholipase A2 were used to analyze the fatty acid distribution. 5. 5. The fatty acid composition of the phosphoglucolipid and its two diglyceride portions was identical. Each of the latter showed a non-random distribution with long-chain fatty acids dominating Position 1 and shorter-chain fatty acids dominating Position 2. 6. 6. Diglucosyl diglyceride, diphosphatidylglycerol and phosphatidylglycerol of S. faecalis showed the same fatty acid composition and distribution pattern as the phosphoglucolipid, suggesting close biosynthetic relationships. 7. 7. Reinvestigation of the phosphoglucolipid from Streptococcus lactis (Fischer W. (1970) Biochem. Biophys. Res. Commun. 41, 731–736) established four acyl groups with the same location as in the phosphoglucolipid of S. faecalis.

Strains of lactic streptococci isolated from samples of raw milk which had developed a malty aroma were subjected to the cultural, physiological, and serological tests commonly employed in the classification of streptococci. None of the strains could be differentiated from Streptococcus lactis by these tests. Resting cells of strains which produced an organoleptically detectable malty aroma when cultured in milk were usually found to possess an active α-ketoacid decarboxylase, indicating the presence of the mechanism responsible for the characteristic aroma production. This decarboxylase activity was either weak or nonexistent in the nonmalty strains, and no activity was detected in known strains of S. lactis, S. cremoris , or S. diacetilactis . The malty strains usually produced higher acidities in milk than did the nonmalty strains, and, in most instances, they developed a granular type of growth sediment in broth, as opposed to a viscid sediment. Many of them gave weakly positive Voges-Proskauer tests in glucose broth with or without added citrate and appeared to be somewhat more resistant to nisin than the nonmalty strains.

SUMMARY: Stable mutant colonies were picked from a parent strain of Streptococcus lactis which gave improved yields of nisin; further improvement could not be produced by means of eolchicine or camphor. Nisin is stable in the culture fluid at acid pH values, but is inactivated by Seitz filtration or heating at pH 6 to 9. A ‘nisinase’ was not found. In the presence of sufficient glucose, growth was increased when the lactic acid formed was neutralized, the concentration of pantothenic acid becoming the limiting factor. Maximum nisin yields were obtained when, during fermentation in the presence of sufficient glucose and pantothenate at 23-30°, the pH was stabilized at c. 6, which is the pH optimum for nisin synthesis. Sodium lactate and nisin are only slightly inhibitory to the organism. Growth comes to an end because of the maximum limiting population density (‘M’-concentration) of cells, and is not due to the exhaustion of food or accumulation of toxic products.

Acid production in milk by lactic streptococci was stimulated by added β-galactosidase. Both glucose and galactose accumulated rapidly in the presence of this enzyme. Glucose accumulation ceased as the culture entered the most rapid period of acid production, whereas galactose accumulation continued. In cultures without added β-galactosidase, a low concentration of galactose accumulated in the milk, whereas glucose was not detected after 2 hr of incubation. Cultures grew and produced acid faster in broth containing glucose rather than galactose or lactose. These observations suggest that the lactic streptococci do not metabolize the lactose in milk efficiently enough to permit optimum acid production and that a phenomenon such as catabolite repression functions to allow for a preferential use of glucose over either galactose or lactose. In addition to providing the culture with a more readily available energy source, it is possible that the culture produced more acidic metabolites as a result of preferentially utilizing the glucose released by the action of the β-galactosidase.

When cells of the anaerobe Streptococcus lactis 7962 are deprived of their normal fermentable energy source, active transport of galactosides is completely abolished although the membrane carriers are still capable of facilitating the equilibration of sugars across the cell membrane. In these nonmetabolizing cells it was possible to test the Mitchell hypothesis of obligatory coupling of proton movement with sugar transport. This hypothesis was supported by alkalinization of the medium observed when thiomethylgalactoside was added to a lightly buffered suspension of S. lactis cells. Conversely, addition of protons resulted in active transport of thiomethylgalactoside. Accumulation of thiomethylgalactoside to a concentration more than 20-times that in the external medium was induced by suddenly exposing cells to a medium at pH 6; no accumulation of thiomethylgalactoside was observed with cells exposed to pH 8. Active transport of thiomethylgalactoside occurred in the absence of energy metabolism when S. lactis cells were treated with valinomycin. This ionophore allowed intracellular K + to flow out, thus imposing a membrane potential (inside negative). This potential resulted in a proton uptake and an associated active transport of galactoside. The membrane potential was measured from the distribution ratio (inside/outside) of K + in the presence of valinomycin. The pH gradient was measured from the distribution ratio of [ 14 C]methylamine. The protonmotive force, calculated from the membrane potential and the pH gradient, was found to be directly related to the accumulation of galactoside, in accordance with the chemiosmotic hypothesis.

Much confusion exists as to the species of streptococci found in milk at different stages in its history, and there is doubt as to whether the identity of the typical streptococcus of sour milk is generally appreciated. The medical bacteriologists usually think of streptococci in milk as indicating a suppuration in the udder or as evidence of some definite contamination, while on the other hand, the dairy bacteriologists usually think of the harmless lactic acid organism. These two points of view are often displayed in attempts to interpret the significance of streptococci in milk.

Streptococcus cremoris HP and Streptococcus lactis 829 were grown in chemostats in tryptone yeast extract broth and in supplemented 2% skim milk medium. In both media, lactose was the limiting nutrient. Cultures were grown at various dilution rates in media poised at constant pH and temperature and also at constant dilution rates in media controlled at different pH levels and temperatures. The effects of the various conditions of growth on production of bacteria, viable counts, and acid-producing activities of cells and on the ability of bacteria to survive subsequent frozen storage were determined. None of the conditions of growth tested had very pronounced effects on the ability of cells to survive or on the inability of cells to retain acid-producing activity after being frozen at −70 °C and stored at −40 °C.

Staphylococcal enterotoxin A production in Blue, Brick, Mozzarella, and Swiss cheeses from milk inoculated with different initial Staphylococcus aureus populations was evaluated. Depending on the type of lactic starter and the strain of S. aureus , enterotoxin was detected under certain conditions in Brick and Swiss cheeses. It was not demonstrated even with higher S. aureus populations (5×10 7 /g) and a complete starter failure in Blue cheese. Minimal S. aureus population associated with presence of detectable enterotoxin was influenced by the environmental conditions in cheese.

SummaryFrozen concentrated cheese starters were prepared from 7Streptococcus cremorisand 2Str. lactisstrains by continuous culture. pH control with NH4OH and Ca(OH)2increased the yield ofStr. cremorisstrains as compared with NaOH, but the resultant cells were smaller and less active. Maximum yields for both species were obtained at a dilution rate of 0·6 and pH 6·3, but withStr. cremoriscell activity was reduced above pH 6·0.Degeneration in continuous culture was observed in the 2Str. lactisstrains, due apparently in one case to the action of a bacteriophage and in the other possibly to selection or to loss of adaptive enzymes.Concentrated starters retained full viability and activity when stored for 10–12 weeks at −110°C, and 94% activity after storage for 4 weeks at −40°C.Cheese made with the concentrated starters ripened normally and was comparable in quality with control cheese made with normal bulk starter.

SummaryEquipment and methods are described for the production, on the laboratory scale, of frozen concentrated cheese starters. A single-stage Porton-type fermenter with a working volume of 3–5 l was used for the continuous culture of theStreptococcus lactisandStr. cremorisstarter strains. The cells grown in a trypsin-digested cheese-whey or trypsin-digested skim-milk, both containing autolysed yeast, were harvested with a Sharples laboratory super-centrifuge, resuspended in skim-milk and layer-frozen in liquid N2. The frozen culture was crushed to a granular free-flowing form facilitating direct addition to and ready dispersion in cheese milk. The cultures were stored at −196°C.

A temperate bacteriophage, designated r 1 t, was inducible from the group N lactic streptococcus, Streptococcus cremoris R 1 , by ultraviolet irradiation or mitomycin C treatment. Induced lysates produced plaques on lawns of three closely related S. cremoris strains, AM 1 , SK 11 , and US 3 . Strain SK 11 was readily lysogenized. S. cremoris AM 1 was the most reliable indicator strain, although the age of the culture used for seeding plates was critical. Zones of lysis but no plaque formation were observed on lawns of nine additional S. cremoris strains. Phage r 1 t could not be detected in filtrates of stationary-phase R 1 cultures and was near the limits of detection in logarithmically growing cultures. Phage levels were still very low (1 plaque-forming unit on AM 1 per 10 induced cells) in induced lysates of R 1 cultures. These low levels of detectable phage may be attributable to an inadequate indicator, lysogenization of the indicator, adsorption of induced phage to cellular debris, concurrent induction of other undetectable phages, or the production of high proportions of defective phages. Electron micrographs of induced R 1 lysates revealed a high incidence of incomplete phage particles, fragments, and ghosts.

MacLeod, Patricia (University of Connecticut, Storrs) and Joseph P. Brown . Fatty acid composition of lipids from Streptococcus cremoris and Streptococcus lactis var. maltigenes . J. Bacteriol. 85: 1056–1060. 1963.—A method for the extraction of lipids from bacterial cells, involving grinding in a ball mill, is described. Cells of Streptococcus lactis var. maltigenes and S. cremoris yielded 5.3 and 5.5%, respectively, of their dry cellular weights as lipid. The lipids of both organisms were separated into three fractions by thin-layer chromatography. Two of these fractions have been classified: one fraction containing phospho- and perhaps glycolipids and the other containing triglycerides. The unidentified fraction was probably either mono- or diglyceride in nature. The fatty acid composition of the total lipid as well as that of each of the separated lipid components from the two lactic strains were compared.

From Streptococcus lactis C2, a fast strain which produced sufficient acid to coagulate sterile nonfat milk in 18 hr at 21C, a slow mutant was isolated which required incubation for at least 48 hr to effect coagulation. Nonfat milk cultures of these two organisms were compared to find causes for the apparent reduced acid production by the slow strain. The two organisms had the same generation time (1.4 hr) in the log phase of growth and produced the same amount of acid per cell. The average viable population of the fast culture after 18 hr at 21C, however, was about four times as great as the slow. Also, the fast culture was four times more proteolytic in nonfat milk than the slow. There was, therefore, a direct relationship between available nitrogen and total growth in milk, reflected by the final amount of acid produced in the cultures.

Dahiya , R. S. (North Carolina State College, Raleigh) and M. L. Speck . Identification of stimulatory factor involved in symbiotic growth of Streptococcus lactis and Streptococcus cremoris . J. Bacteriol. 85: 585–589. 1963.—Single-strain cultures of Streptococcus lactis and S. cremoris isolated from a commercial starter culture showed symbiotic growth in milk. This study dealt with identification of the main component responsible for high acid production resulting from the combined growth of these cultures. Paper chromatography and bioautography were adapted to isolate the main stimulatory factor excreted by the slower-growing culture ( S. lactis ) in the culture media. It was identified as adenine on the basis of ultraviolet absorption spectra and paper chromatographic R F value. Pure adenine possessed the same properties and was also stimulatory to the faster-growing culture ( S. cremoris ) when added to milk.

β-D-Phosphogalactoside galactohydrolase (β-Pgal) was examined in a number of lactic streptococci by use of the chromogenic substrate o -nitrophenyl-β-D-galactopyranoside-6-phosphate. Specific activity of β-Pgal ranged from 0.563 units/mg of protein in Streptococcus lactis UN, to 0.120 in S. diacetilactics 18-16. Essentially no β-D-galactoside galactohydrolase (β-gal) was found in these organisms when o -nitrophenyl-β-D-galactopyranoside served as the chromogenic substrate. S. lactis 7962 was the one exception found. This organism contained rather high levels of β-gal, and very little β-Pgal could be detected. β-Pgal activity was examined in streptococci that differed widely in both their proteolytic ability and rates of lactic acid production during growth in milk. Differences in proteolytic ability did not influence β-Pgal synthesis; also, the rate of lactic acid production was independent of the level of β-Pgal present in the cell, since the rate of lactic acid production could be increased approximately fourfold without changing the amount of β-Pgal present in the cell. Various carbohydrates were tested as potential inducers of the enzyme. Although galactose, either as the free sugar or combined with glucose in lactose, was the only inducer, noninducing sugars such as mannose or glucose showed some ability to cause fluctuations in the basal level of β-Pgal. Cells growing in mannose or glucose exhibited about 30% of the maximal enzyme levels found in cells growing in lactose or galactose. No gratuitous inducers were found.

Five plasmids, distinguishable by their molecular weights (10 6 , 2 × 10 6 , 5 × 10 6 , 10 7 , 3 × 10 7 , respectively) were isolated from Streptococcus lactis C2. A spontaneous proteinase-negative derivative of this strain lacked the 10 7 plasmid.

The viability of freeze-dried cells of Streptococcus lactis C-2 was studied by suspending washed cells in reconstituted skim milk and in solutions of ascorbic acid, thiourea and ammonium chloride used alone or in various combinations. Low cell survival was observed with the use of reconstituted skim milk. Fortification of this menstruum with ascorbic acid and thiourea gave maximum survival of freeze-dried cells.

Summary The lipids of the two-day cultures of S. lactis were extracted and fractionated. The non-polar lipids prevailed. The absence of lactobacillic acid was found to be typical for the nisin-producing strains.

Streptococcus lactis C2 has been used extensively by many laboratories in studies on the metabolism of lactic streptococci. By using ultraviolet irradiation as the inducing agent, this organism was shown to release a small bacteriophage, indicating that it is a lysogenic strain. The induced phage had a head approximately 40 nm in diameter and a tail length and width of about 180 and 6 nm, respectively. A structure resembling a collar was observed. Attempts to isolate a sensitive indicator strain for the virus were unsuccessful.

Bestatigung fruherer Feststellungen, dass Citral eine antibakterielle Aktivitat besitzt. Da Neral und Geranial (allein oder zusammen) jedoch wesentlich weniger aktiv sind, muss fur die Citralaktivitat eine bisher nicht identifizierte Substanz verantwortlich sein.

Group N streptococci possess enzymes for the metabolism of d -galactose through both the d -galactose 1-phosphate pathway (Leloir pathway) and the d -tagatose 6-phosphate pathway.

SUMMARY: Some strains of Streptococcus cremoris are slightly more heat resistant than the majority. After growth in milk at 37° for 5 hr. some of them show under the microscope involution forms which are sufficiently characteristic to serve as a useful means of identification.