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Phosphate/hexose 6-phosphate antiport in Streptococcus lactis.

After growth in appropriate media, resting cells of Streptococcus lactis 7962 showed a rapid exchange between external and internal pools of inorganic phosphate. This exchange was not found in other strains of S. lactis (ML3, 133, or K1) or in Streptococcus faecalis. Phosphate exchange in S. lactis 7962 did not require other anions or cations in the assay medium, nor was phosphate influx affected by the membrane potential and pH gradient formed during glycolysis. Thus, the exchange reaction was independent of known ionic drivers (H+, Na+, OH-, etc.). Experiments testing inhibitions of phosphate entry suggested that alternative substrates for exchange included arsenate, as well as the 6-phosphates of glucose, 2-deoxyglucose, fructose, mannose, or glucosamine, and direct studies with 2-deoxyglucose 6-phosphate verified that resting cells could accumulate this sugar phosphate to levels expected for exchange with internal phosphate. Two other observations supported the idea of an exchange between phosphate and sugar phosphate. First, early addition of the heterologous substrate blocked entry of the test compound, whereas later addition caused efflux of preaccumulated material. Second, expression of phosphate exchange and 2-deoxyglucose 6-phosphate transport varied in parallel. Both activities were found at high levels after growth in medium supplemented with rhamnose or arabinose, at intermediate levels with addition of galactose, and at low levels after growth with glucose, fructose, or mannose. We conclude that these findings describe a novel anion antiporter that mediates the exchange of phosphate (arsenate) and sugar 6-phosphates.

Anions↗

IDENTIFICATION OF GROWTH STIMULANTS FOR STREPTOCOCCUS LACTIS.

Koburger, J. A. (North Carolina State College, Raleigh), M. L. Speck, and L. W. Aurand. Identification of growth stimulants for Streptococcus lactis. J. Bacteriol. 85:1051-1055. 1963-The growth of Streptococcus lactis in milk was accelerated by pancreas extract, and three distinct components that were detected by bioautography were isolated and identified. These were purified by precipitation with silver ions, and their silver salts were regenerated in acidic solution. After adsorption and elution from characoal, contaminating alpha-amino compounds were destroyed by treatment with ninhydrin. Final purification was by chromatography. Identification of the purified fractions was by paper chromatography, and by ultraviolet and infrared spectra. The active compounds were found to be inosine, hypoxanthine, and adenine.

Acids↗

BETA-GALACTOSIDASE OF STREPTOCOCCUS LACTIS.

Citti, J. E. (Oregon State University, Corvallis), W. E. Sandine, and P. R. Elliker. beta-Galactosidase of Streptococcus lactis. J. Bacteriol. 89:937-942. 1965.-Synthesis of beta-galactosidase by several strains of Streptococcus lactis was induced by lactose. The rate of hydrolysis of o-nitrophenyl-beta-d-galactopyranoside was used to measure enzyme activity. The enzyme of all but one strain was unstable when whole cells were sonic-treated or treated with toluene; the enzyme of one strain of S. lactis was stable to these treatments, which resulted in at least a fivefold increase in activity over that found in whole cells. The optimal assay conditions for toluene-treated cells of this strain involved incubation at 37 C in pH 7.0 sodium phosphate buffer. Lactose was the most effective inducer of enzyme synthesis. Methyl-beta-d-thiogalactopyranoside, isopropyl-beta-d-thiogalactopyranoside, and galactose were also inducers of the enzyme, but were not as effective as lactose. Melibiose, maltose, and calcium lactobionate were poor inducers of enzyme synthesis. Exogenously supplied glucose repressed enzyme synthesis. The means of control of induced beta-galactosidase synthesis in S. lactis was similar to that in Escherichia coli.

Disaccharides↗

Effect of low pH on thiomethyl-beta-D-galactoside uptake by Streptococcus lactis.

Maximal beta-galactosidase activity in Streptococcus lactis was obtained at pH 7, but the maximal rate of thiomethyl-beta-d-galactoside uptake was observed at pH 3.6 to 4. It is concluded that the decrease in beta-galactosidase activity in intact cells at lowered pH is not due to diminished transport of beta-galactoside.

Glycosides↗

Acyldiglucosyldiacylglycerol-containing lipoteichoic acid with a poly(3-O-galabiosyl-2-O-galactosyl-sn-glycero-1-phosphate) chain from Streptococcus lactis Kiel 42172.

The lipoteichoic acid of Streptococcus lactis Kiel 42172 was isolated. The lipid portions were released by HF and were established to be 3-O-[O-alpha-D-galactopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl]-2-O-alpha-D-galactopyranosyl-sn-glycero-1-phosphate, they are joined by phosphodiester bonds nosyl)]glycerol. The repeating units of the hydrophilic chain were established to be 3-O-[O-alpha-D-galactopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl]-2-O-alpha-D-galactopyranosyl-sn-glycero-1-phosphate; they are joined by phosphodiester bonds at carbon atom 6 of the galabiosyl residues. The innermost unit is linked to the glycolipid by a phosphodiester presumably at C-6 of the outer glucosyl moiety. The hydrophilic chain is 7.4--11.8 units in length, measuring 12--19 nm is extended conformation. The content of 2.7--2.96 acyl groups per 2 glucosyl residues indicates that 70--96% of the glycolipid consists of acyldiglucosyldiacylglycerol. The novel poly(glycosylgly-cerophosphate) structure provided for the first time the oplipoteichoic acids are the sn-1 isomer which has previously been suggested from biosynthetic studies (Glaser, L., & Lindsay, B. (1974) Biochem. Biophys. Res. Commun. 59, 1131--1136).

Carbohydrates↗

Proton-coupled accumulation of galactoside in Streptococcus lactis 7962.

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.

Biological Transport, Active↗

Arginine transport in Streptococcus lactis is catalyzed by a cationic exchanger.

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway to ornithine, CO2, NH3, and ATP. The translocation of arginine and ornithine has been studied using membrane vesicles of galactose/arginine-grown cells of S. lactis fused with cytochrome c oxidase proteoliposomes by the freeze/thaw--sonication procedure earlier described. In the presence of reduced cytochrome c the fused membranes rapidly accumulate ornithine. Addition of arginine releases accumulated ornithine. Rapid uncoupler-insensitive exchange between external arginine and internal ornithine is seen at rates that are at least 60-fold higher than the rate of protonmotive force-driven arginine translocation. This arginine:ornithine exchange activity was reconstituted in proteoliposomes after solubilization of S. lactis membranes with octyl beta-D-glucopyranoside. These proteoliposomes catalyze a one-to-one exchange between arginine and ornithine. The arginine:ornithine exchange system is the first exchange system for cationic metabolites found in bacteria. Translocation of arginine via this system does not require metabolic energy obtained by arginine metabolism.

Antiporters↗

A method for genetic transformation of nonprotoplasted Streptococcus lactis.

Plasmid transformation of whole cells of Streptococcus lactis LM0230 was demonstrated. The procedure required polyethylene glycol and incubation in hypertonic media, but did not require enzymatic cell wall digestion. Conditions were optimized, yielding 5 X 10(5) transformants per micrograms of pSA3 DNA. Variables tested for effect on transformation efficiency included molecular weight, concentration, and pH of polyethylene glycol; cell density; plating media; DNA concentration; heat shock; and incubation of cells in hypertonic buffer. DNAs transformed included pSA3, pVA856, pTV1, and c2 phi. Transformation from DNA-DNA ligation mixes, with DNA not purified through density gradients, and with previously frozen cells was also achieved. The method described here for transformation of nonprotoplasted cells of LM0230 is unique, and to date has not been applied successfully to other lactic acid bacteria.

Cloning, Molecular↗

Cloning and expression of an alpha-acetolactate decarboxylase gene from Streptococcus lactis subsp. diacetylactis in Escherichia coli.

The Streptococcus lactis gene coding for alpha-acetolactate decarboxylase (ADC) was cloned in Escherichia coli. Subsequent subcloning in E. coli showed that the ADC gene was located within a 1.3-kilobase DNA fragment. The ADC gene was controlled by its own promoter. Gas chromatography showed that S. lactis and the transformed E. coli strains produced the two optical isomers of acetoin in different ratios.

Acetoin↗

Proton motive force during growth of Streptococcus lactis cells.

Experiments with the aerotolerant anaerobe Streptococcus lactis provide the opportunity for determining the proton motive force (Deltap) in dividing cells. The two components of Deltap, DeltaPsi (the transmembrane potential) and DeltapH (the chemical gradient of H(+)), were determined by the accumulation of radiolabeled tetraphenylphosphonium (TPP(+)) and benzoate ions. The DeltaPsi was calibrated with the K(+) diffusion potential in starved, valinomycin-treated cells. With resting, glycolyzing cells, the Deltap was measured also by the accumulation of the non-metabolizable sugar thiomethyl-beta-galactoside (TMG). In resting cells the Deltap, calculated either by adding DeltaPsi and ZDeltapH or from the levels of TMG, was relatively constant between pH 5 to 7, decreasing from 160 to 150 mV and decreasing further to 100 mV at pH 8.0. With the TPP(+) probe for DeltaPsi, we confirmed our previous finding that the K(+) ions dissipate DeltaPsi and increase DeltapH, whereas Na(+) ions have little effect on DeltaPsi and no effect on DeltapH. [(3)H]TPP(+) and [(14)C]benzoate were added during exponential phase to S. lactis cells growing at pH 5 to 7 at 28 degrees C in a defined medium with glucose as energy source. As with resting cells, the DeltapH and DeltaPsi were dependent on the pH of the medium. At pH 5.1, the DeltapH was equivalent to 60 mV (alkaline inside) and decreased to 25 mV at pH 6.8. The DeltaPsi increased from 83 mV (negative inside) at pH 5.1 to 108 mV at pH 6.8. The Deltap, therefore, was fairly constant between pH 5 and 7, decreasing from 143 to 133 mV. The values for Deltap in growing cells, just as in resting cells, are consistent with a system in which the net efflux of H(+) ions is effected by a membrane-bound adenosine triphosphatase and glycolytically generated adenosine triphosphate. The data suggest that in both growing and resting cells the pH of the medium and its K(+) concentration are the two principal factors that determine the relative contribution of DeltapH and DeltaPsi to the proton motive force.

Hydrogen↗

Uptake and metabolism of sucrose by Streptococcus lactis.

Transport and metabolism of sucrose in Streptococcus lactis K1 have been examined. Starved cells of S. lactis K1 grown previously on sucrose accumulated [14C]sucrose by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (sucrose-PTS; Km, 22 microM; Vmax, 191 mumol transported min-1 g of dry weight of cells-1). The product of group translocation was sucrose 6-phosphate (6-O-phosphoryl-D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside). A specific sucrose 6-phosphate hydrolase was identified which cleaved the disaccharide phosphate (Km, 0.10 mM) to glucose 6-phosphate and fructose. The enzyme did not cleave sucrose 6'-phosphate(D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside-6'-phosphate). Extracts prepared from sucrose-grown cells also contained an ATP-dependent mannofructokinase which catalyzed the conversion of fructose to fructose 6-phosphate (Km, 0.33 mM). The sucrose-PTS and sucrose 6-phosphate hydrolase activities were coordinately induced during growth on sucrose. Mannofructokinase appeared to be regulated independently of the sucrose-PTS and sucrose 6-phosphate hydrolase, since expression also occurred when S. lactis K1 was grown on non-PTS sugars. Expression of the mannofructokinase may be negatively regulated by a component (or a derivative) of the PTS.

Biological Transport↗

Injury and death of Streptococcus lactis due to freezing and frozen storage.

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a "restricted" lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.

Agar↗

Damage to Streptococcus lactis resulting from growth at low pH.

Harvey, R. J. (The Dairy Research Institute, Palmerston North, New Zealand). Damage to Streptococcus lactis resulting from growth at low pH. J. Bacteriol. 90:1330-1336. 1965.-Growth of Streptococcus lactis below pH 5.0 results in damage to the cells. A period of growth below pH 5.0 is required before this damage is expressed, and a period of growth above pH 5.0 is required to correct the damage. Part of the damage consists of a reduction in the specific activities of a number of enzymes. At the same time, the differential rate of total protein synthesis is unchanged. During recovery from growth at low pH, the enzymes subject to damage are formed at a differential rate which is higher than normal until the normal specific activities are restored. The growth rate characteristic of the new pH is then resumed. The results are consistent with either of two mechanisms of damage. Growth at low pH could result either in direct inactivation of a number of enzymes, or in loss of control of the differential rates of synthesis of individual enzymes.

Hydrogen-Ion Concentration↗

[Physiological characteristics of immobilized cells of Streptococcus lactis, Moscow State University strain].

Immobilized cells of Streptococcus lactis, strain MSU, synthesized nisin by 20--30 per cent of the possible maximum antibiotic production level. The immobilized cells of the Streptococcus synthesized nisin by 40--50 per cent probably at the expense of the cell nutrient pools and by 50--60 per cent at the expense of the medium nutrient consumption. The electron microphotographs showed that the cells of Str. lactis, strain MSU, preserved their usual appearance in the granules of polyacrylamide gel 24 hours after immobilization. In 3 weeks there were observed structural changes in the cell wall and inner membranes resulting in the cell lysis after presence in the granules for 3 months.

Bacteriological Techniques↗

Quantity and fatty acid composition of lipid extracted from cells of Streptococcus lactis.

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.

Chromatography, Gas↗

Chemical nature of malty flavor and aroma produced by Streptococcus lactis var. maltigenes.

Mature skim milk cultures of Streptococcus lactis var. maltigenes were steam distilled at low temperature under reduced pressure. Ethyl ether extracts were prepared from the distillates and analyzed by gas-liquid chromatography and mass spectrometry. Twenty of 31 components detected in the culture distillates were identified positively and 11 tentatively, whereas 10 of 19 components detected in the heated skim milk control were identified positively and 9 tentatively. Among components detected in the culture distillate, but not detected in the heated skim milk distillate, and which have not been previously identified in milk cultures of the organism were phenylacetaldehyde and phenethanol. Quantitative analyses of the volatiles entrained from milk cultures of several strains of S. lactis var. maltigenes revealed a probable relationship between variation in the character of the aroma of the cultures and the alcohol/aldehyde ratio.

Acids↗

Purification and properties of Streptococcus lactis beta-galactosidase.

beta-Galactosidase of Streptococcus lactis 7962 was partially purified, and its properties were studied. Enzyme from only this strain of numerous lactic streptococci tested was stable in cell exudates prepared by various means. Cell-free extracts of the 7962 strain were prepared by sonic treatment of washed cells previously grown in presence of lactose to fully induce enzyme synthesis. Protamine sulfate precipitation of the nucleic acids and ammonium sulfate precipitation of protein were used for partial purification of the enzyme. The resulting enzyme, when resuspended in cold (5 C) phosphate buffer, was extremely labile. However, ammonium sulfate in high concentrations (0.85 m) stabilized and stimulated beta-galactosidase activity. Sephadex G-200 gel filtration was used to achieve further purification and to monitor homogeneity of the enzyme. Separation of the beta-galactosidase in buffer at 5 C yielded an enzyme elution pattern showing two peaks of activity. However, addition of the enzyme solution in 0.85 m ammonium sulfate to the column equilibrated with the same salt concentration yielded only one peak of enzyme activity. The data suggested that the native enzyme was dissociating into active subunits which were stabilized in the presence of the ammonium sulfate.

Chromatography, Gel↗