Comparative acid tolerances and inhibitor sensitivities of isolated F-ATPases of oral lactic acid bacteria.

pH activity profiles and inhibitor sensitivities were compared for membrane ATPases isolated from three oral lactic acid bacteria, Lactobacillus casei ATCC 4646, Streptococcus mutans GS-5, and Streptococcus sanguis NCTC 10904, with, respectively, high, moderate, and low levels of acid tolerance. Membranes containing F1F0 ATPases were isolated by means of salt lysis of cells treated with muralytic enzymes. Membrane-free F1F0 complexes were then isolated from membranes by detergent extraction with Triton X-100 or octylglucoside. Finally, F1 complexes free of the proton-conducting F0 sector were obtained by washing membranes with buffers of low ionic strength. The pH activity profiles of the membrane-associated enzymes reflected the general acid tolerances of the organisms from which they were isolated; for example, pH optima were approximately 5.5, 6.0, and 7.0, respectively, for enzymes from L. casei, S. mutans, and S. sanguis. Roughly similar profiles were found for membrane-free F1F0 complexes, which were stabilized by phospholipids against loss of activity during storage. However, profiles for F1 enzymes were distinctly narrower, indicating that association with F0 and possibly other membrane components enhanced tolerance to both acid and alkaline media. All of the enzymes were found to have similar sensitivities to Al-F complexes, but only F1F0 enzymes were highly sensitive to dicyclohexylcarbodiimide. The procedures described for isolation of membrane-free F1F0 forms of the enzymes from oral lactic acid bacteria will be of use in future studies of the characteristics of the enzymes, especially in studies with liposomes.     

Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.     

Environmental pH as a factor in the competition between strains of the oral streptococci Streptococcus mutans, S. sanguis, and "S. mitior" growing in continuous culture

Strains of Streptococcus mutans (biotype 1), Streptococcus sanguis, and Streptococcus mitior have been grown in mixed continuous culture in a semidefined medium under glucose limitation at a growth rate of D = 0.1 h-1. The effect of varying the environmental pH on the proportions of the different populations within the community has been determined. Initially the populations were allowed to reach steady state at pH 7.0 when S. sanguis was dominant with S. mutans and "S. mitior" maintaining similar populations. The medium pH was then lowered in steps of 0.5 pH units from pH 7.0 to 4.5, and the community was grown at each step for at least 15 generations. Viable counts of each species were made at 24-h intervals. The population ratios established at pH 7.0 remained relatively stable when the environmental pH was set at pH 6.5. However, after the medium pH was lowered to 6.0 (days 18-27), the population of S. mutans began to increase and the S. mitior population began to decline. A further change was seen at pH 5.5 (days 27-34) when S. mutans became dominant, S. sanguis declined, and S. mitior was not detectable. At pH 4.5, both S. mutans and S. sanguis were reduced in numbers, but survived until the experimental run was terminated (44 days). Samples of culture fluid were taken throughout the experiment and analyzed for the presence of the acid products of glucose metabolism. The amounts of lactic acid produced by the community increased as the environmental pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of lowering the pH on the composition and metabolism of a community of nine oral bacteria grown in a chemostat

Nine oral bacteria, associated with both healthy and diseased sites in the mouth, were grown at D = 0.05 h-1 (mean generation time 13.9 h) in a glucose-limited chemostat. After an initial period of steady-state growth at pH 7.0, pH control was discontinued. The pH then decreased until it stabilized at pH 4.1 after 9 d (16 generations), while the Eh rose from -165 mV to +160 mV. The lowering in pH resulted in the composition and metabolism of the flora being altered and in increased bacterial aggregation. At pH 7.0, 'Streptococcus mitior', Veillonella alcalescens and S. sanguis were most numerous while at pH 4.1 the counts of all bacteria fell except for Lactobacillus casei, which became predominant. The proportions of S. mutans within the community also increased while S. sanguis was recovered only occasionally and Bacteroides intermedius was not detected below pH 4.6. The survival at pH 4.1 of several other species would not have been predicted from earlier pure culture studies. Relative to pH 7.0, the community growing at pH 4.1 produced more lactic acid, washed cells had a greater glycolytic activity over a wider pH range but amino acid metabolism decreased. In general, when pH control was restored, so were the original patterns of metabolism and bacterial counts, except for B. intermedius, which was still not detected. The inverse relationship between S. sanguis and S. mutans, and the increase in proportions of L. casei and S. mutans during growth in a low pH environment parallel observations made in vivo and suggest that the chemostat can be used as a model for microbial behaviour in dental plaque.     

Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.     

Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance.

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.     

Low pH-induced membrane fatty acid alterations in oral bacteria

Four oral bacterial strains, of which two are considered aciduric and two are considered acid-sensitive, were grown under glucose-limiting conditions in chemostats to determine whether their membrane fatty acid profiles were altered in response to environmental acidification. Streptococcus gordonii DL1, as well as the aciduric strains S. salivarius 57.I, and Lactobacillus casei 4646 increased the levels of mono-unsaturated membrane fatty acids. The non-aciduric strain S. sanguis 10904 did not alter its membrane composition in response to pH values examined here. Thus, in response to low pH, aciduric oral bacteria alter their membrane composition to contain increased levels of long-chained, mono-unsaturated fatty acids. This suggests that membrane fatty acid adaptation is a common mechanism utilized by bacteria to withstand environmental stress.     

Reconstitution of ATP-dependent calcium transport from streptococci

Membrane vesicles of three streptococcal strains (Streptococcus faecalis, Streptococcus lactis, and Streptococcus sanguis) were extracted with octyl-beta-D-glucoside in the presence of Escherichia coli lipid and glycerol. For reconstitution, the detergent extract was mixed with bath-sonicated E. coli lipid, in the presence of octyl-beta-D-glucoside, and proteoliposomes were formed by a 25-fold dilution. ATP-dependent calcium accumulation by proteoliposomes was comparable to that found in parent vesicles. Recovery of this calcium transport activity was dependent on the inclusion of an osmolyte protein stabilant (glycerol, etc.) during solubilization. The properties of ATP-driven calcium transport were studied in the reconstituted system. In proteoliposomes, ATP-linked calcium accumulation was not affected by the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or by the ionophores, valinomycin and nigericin, in the presence of potassium, or by N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. On the other hand, calcium transport was completely blocked by micromolar levels of orthovanadate; half-maximal inhibitions were observed at 0.4, 4, and 4 microM vanadate, for S. faecalis, S. lactis, and S. sanguis, respectively. This marked sensitivity to orthovanadate suggests operation of an E1E2-type ion-motive pump. These data demonstrate that, in a reconstituted system, calcium transport is not linked to an ATP-dependent proton circulation via the F0F1-ATPase, but rather is driven by a calcium-translocating ATPase. Thus, calcium extrusion from the cytosol of enteric, lactic acid, or oral streptococci is mediated by an ATP-linked process analogous to the ion-motive ATPases of eukaryotic membranes.     

Lactic acid bacteria from healthy oral cavity of Thai volunteers: inhibition of oral pathogens

The aims of the present study were to screen and characterize the antimicrobial lactic acid bacteria which were isolated from healthy oral cavities of Thai volunteers, and to characterize their inhibiting substances. Among 3790 isolates (suspected to be lactic acid bacteria) from 130 volunteers, five showed an appreciable effect against Sarcina lutea ATCC 9341, Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Streptococcus mutans DTMU 1, Strep. salivarius DTMU 1, Strep. sanguis DTMU 1, Candida albicans ATCC 13803 and C. albicans DTMU 2, as well as the oral pathogens. These antimicrobial isolates included L17 and N14 which showed the antibacterial activity, D14 which showed the anticandidal activity, and D6 and N8 which showed both the antibacterial and anticandidal activities. The isolates were later found to be facultative anaerobic, Gram-positive, non-spore-forming, non-capsule-forming and catalase-negative bacilli. They could utilize casein but could not hydrolyse starch, and they produced hydrogen peroxide and bacteriocins. Their antimicrobial potentials were found to be affected by pH, catalase, proteolytic enzymes and temperature. The activity was partially inactivated after catalase treatment, significantly declined at pH > or =9.0 or after trypsin and pepsin treatments, and also reduced after heating at > or =100 degrees C. However, the antimicrobial activity of these five isolates was somewhat resistant to heat. When the isolates were tested for their antimicrobial sensitivity, they were shown to be sensitive to a number of antimicrobial agents. The final identification revealed that D6, D14 and N14 were Lactobacillus paracasei subsp. paracasei, and L17 and N8 were Lact. rhamnosus.     

Autolysis in strains of viridans streptococci.

Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.     

苹果多酚作为微生态调节剂对窝沟致龋菌调节作用的研究

目的初步探讨苹果多酚作微生态调节剂抑制变形链球菌,扶持血链球菌生长的实验研究,为龋病的防治提供一定的依据。方法采用抑菌环实验观察苹果多酚对变形链球菌和血链球菌生长的影响,检测其对变形链球菌产酸的变化,并通过扫描电镜观察对变形链球菌在玻片粘附情况的影响。结果苹果多酚能够明显抑制变形链球菌的生长,阻止其对玻片的粘附,降低其产酸能力,但对血链球菌生长没有明显的抑制作用。对变形链球菌与血链球菌的影响比较差异有统计学意义。结论苹果多酚具有调整窝沟致龋菌的作用,进一步研究很可能是一良好的窝沟菌群的微生态调节剂。     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Anticariogenic activity of macelignan isolated from Myristica fragrans (nutmeg) against Streptococcus mutans

The occurrence of dental caries is mainly associated with oral pathogens, especially cariogenic Streptococcus mutans. Preliminary antibacterial screening revealed that the extract of Myristica fragrans, widely cultivated for the spice and flavor of foods, possessed strong inhibitory activity against S. mutans. The anticariogenic compound was successfully isolated from the methanol extract of M. fragrans by repeated silica gel chromatography, and its structure was identified as macelignan by instrumental analysis using 1D-NMR, 2D-NMR and EI-MS. The minimum inhibitory concentration (MIC) of macelignan against S. mutans was 3.9 microg/ml, which was much lower than those of other natural anticariogenic agents such as 15.6 microg/ml of sanguinarine, 250 microg/ml of eucalyptol, 500 microg/ml of menthol and thymol, and 1000 microg/ml of methyl salicylate. Macelignan also possessed preferential activity against other oral microorganisms such as Streptococcus sobrinus, Streptococcus salivarius, Streptococcus sanguis, Lactobacillus acidophilus and Lactobacillus casei in the MIC range of 2-31.3 microg/ml. In particular, the bactericidal test showed that macelignan, at a concentration of 20 microg/ml, completely inactivated S. mutans in 1 min. The specific activity and fast-effectiveness of macelignan against oral bacteria strongly suggest that it could be employed as a natural antibacterial agent in functional foods or oral care products.     

Triclosan inhibition of membrane enzymes and glycolysis of Streptococcus mutans in suspensions and biofilms

Triclosan was found to be a potent inhibitor of the F(H+)-ATPase of the oral pathogen Streptococcus mutans and to increase proton permeabilities of intact cells. Moreover, it acted additively with weak-acid transmembrane proton carriers, such as fluoride or sorbate, to sensitize glycolysis to acid inhibition. Even at neutral pH, triclosan could inhibit glycolysis more directly as an irreversible inhibitor of the glycolytic enzymes pyruvate kinase, lactic dehydro genase, aldolase, and the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Cell glycolysis in suspensions or biofilms was inhibited in a pH-dependent manner by triclosan at a concentration of about 0.1 mmol/L at pH 7, approximately the lethal concentration for S. mutans cells in suspensions. Cells in intact biofilms were almost as sensitive to triclosan inhibition of glycolysis as were cells in suspensions but were more resistant to killing. Targets for irreversible inhibition of glycolysis included the PTS and cytoplasmic enzymes, specifically pyruvate kinase, lactic dehydrogenase, and to a lesser extent, aldolase. General conclusions are that triclosan is a multi-target inhibitor for mutans streptococci, which lack a triclosan-sensitive FabI enoyl-ACP reductase, and that inhibition of glycolysis in dental plaque biofilms, in which triclosan is retained after initial or repeated exposure, would reduce cariogenicity.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Membrane ATPases and acid tolerance of Actinomyces viscosus and Lactobacillus casei.

Lactobacillus casei ATCC 4646 and Actinomyces viscosus OMZ105E were found to differ markedly in acid tolerance. For example, pH profiles for glycolysis of intact cells in dense suspensions indicated that glycolysis by L. casei had an optimal pH of about 6.0 and that glucose degradation was reduced by 50% at a pH of 4.2. Comparable values for A. viscosus cells were at pHs of about 7.0 and 5.6. The difference in acid tolerance appeared to depend mainly on membrane physiology, and the addition of 40 microM gramicidin to cell suspensions increased the sensitivity of the glycolytic system by as much as 1.5 pH units for L. casei and up to 0.5 pH unit for A. viscosus. L. casei cells were inherently somewhat more resistant to severe acid damage than were A. viscosus cells, in that Mg release from L. casei cells in medium with a pH of 3.0 occurred only after a lag of some 4 h, compared with rapid release from A. viscosus cells. However, the major differences pertinent to the physiology of the organisms appeared to be related to proton-translocating ATPases. Isolated membranes of L. casei had about 3.29 U of ATPase per mg of protein, compared with only about 0.06 U per mg of protein for those of A. viscosus. Moreover, the ATPase of L. casei had a pH optimum for hydrolytic activity of about 5, compared with an optimal pH of about 7 for that of A. viscosus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane ATPases and acid tolerance of Actinomyces viscosus and Lactobacillus casei

Lactobacillus casei ATCC 4646 and Actinomyces viscosus OMZ105E were found to differ markedly in acid tolerance. For example, pH profiles for glycolysis of intact cells in dense suspensions indicated that glycolysis by L. casei had an optimal pH of about 6.0 and that glucose degradation was reduced by 50% at a pH of 4.2. Comparable values for A. viscosus cells were at pHs of about 7.0 and 5.6. The difference in acid tolerance appeared to depend mainly on membrane physiology, and the addition of 40 microM gramicidin to cell suspensions increased the sensitivity of the glycolytic system by as much as 1.5 pH units for L. casei and up to 0.5 pH unit for A. viscosus. L. casei cells were inherently somewhat more resistant to severe acid damage than were A. viscosus cells, in that Mg release from L. casei cells in medium with a pH of 3.0 occurred only after a lag of some 4 h, compared with rapid release from A. viscosus cells. However, the major differences pertinent to the physiology of the organisms appeared to be related to proton-translocating ATPases. Isolated membranes of L. casei had about 3.29 U of ATPase per mg of protein, compared with only about 0.06 U per mg of protein for those of A. viscosus. Moreover, the ATPase of L. casei had a pH optimum for hydrolytic activity of about 5, compared with an optimal pH of about 7 for that of A. viscosus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uncommon pathways of metabolism among lactic acid bacteria

A small number of lactic acid bacteria possess the ability to derive energy from organic molecules not utilized by the vast majority of representatives of this large group of microorganisms. Thus, strains of Lactobacillus casei and enterococci readily grow at the expense of substrates such as gluconate, malate and pentitols. Transport of gluconate and pentitols is catalysed by phosphotransferase systems unique to these bacteria. Similarly, the initial steps in pentitol dissimilation are mediated by enzymes found only in Lb. casei and Streptococcus avium.     

Uncommon pathways of metabolism among lactic acid bacteria

Abstract A small number of lactic acid bacteria possess the ability to derive energy from organic molecules not utilized by the vast majority of representatives of this large group of microorganisms. Thus, strains of Lactobacillus casei and enterococci readily grow at the expense of substrates such as gluconate, malate and pentitols. Transport of gluconate and pentitols is catalysed by phosphotransferase systems unique to these bacteria. Similarly, the initial steps in pentitol dissimilation are mediated by enzymes found only in Lb. casei and Streptococcus avium .     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.