Archs WOI thi.
Vol. ?I. up. 677 lo 683. Pergamon
Press 1976 Prmted m Great Brltam
LACTATE DEGRADATION BY A STRAIN OF NEISXERIA ISOLATED FROM HUMAN DENTAL PLAQUE ETSURO HOSHINO, T. YAMADA and S. ARAYA Laboratory of Oral Biochemistry, Tohoku University, School of Dentistry, Sendai, 980 Japan Summary--An oral Neisseriu strain Ne-15 degraded lactate under aerobic conditions. Most of the lacr ate, both the L( + ) and D( - ) forms, was converted via pyruvate to acetate, and it seems that ATP may be generated through this pathway. Part of the lactate was converted to acetate directly. A small portion of pyruvate was further metabolized by the way of the citric acid cycle after CO, fixation or after conversion to acetyl-CoA, and was utilized for synthesis cf cell components. The addition of the Neisseria in a mixed incubation with Streptococcus mu~ans caused no change in glucose utilization but a reduced lactate concentration.
INTRODUCTION Dental plaque de;;rades glucose not only to lactate but also to volatile acids and under aerobic conditions rapid destruction of lactic acid occurs at neutral pH (Muntz, 1943). Lactate may be converted to some weak and volatile acids which are predominant in ‘resting plaque’ irr ~iuo (Geddes, 1972; 1975). Veillonella, one of the microorganisms which degrade lactate and form volatile acids, reduces the cariogenic activity of Strrptococcus mutans in rats (Mikx et u1., 1972). The concomitant presence of Neisseria in denta plaque (Hemmens et al., 1946; Morris, 1954; Ritz, 1967 and 1969; Slidaway, 1969) and their ability to metabolize lactate (Berger, 1962) may imply that these microorganisms take a part in the degradation of lactate in dental plaque and play an important role in the microbial ecology of the plaque. MATERIALSAND
METHODS
M icrovrganisnzs Neisseria strair 15 (Ne-15) isolated from human dental plaque was obtained from Prof. I. Takazoe, Tokyo Dental College, and cultured in shallow layer (about 1Omm deep) of trypticase soy broth (BBL, Cockeysville) at 3 5°C with shaking. Each culture was Gram-stained, tested for the presence of oxidase (Kovacs, 1956) and for catalase. Ne-15 was identified as a saccharolytic Neisseria similar to that isolated from the dental plaque of children in England (G. H. Bowden, personal communication). Streptococcus mutans PK 1 (Gibbons et a/., 1966) was supplied by Prof. W. Kondo, School of Dentistry, Niigata University, Niigata, and was cultured in Gibbons and Nygaard’s (1968) medium supplemented with 2 per cent (w/v) glucose and 0.1 per cent (w/v) yeast extract. The purity was checked on blood agar. Both organisms were harvested during their logarithmic growth phase by centrifugation and washed three times with 40 mM-potassium phosphate buffer, pH 7.0, at 4°C and suspended in the same buffer. The dry weight of the cells was estimated from the turbidity of the suspension at 660nm.
The same cells of Ne-15 were subjected to enzyme assay. A cell-free extract of Ne-15 was prepared by the method described by Yamada and Araya (1975). Characterization
I$ Ne-15
Fermentation of glucose, fructose, maltose or sucrose was tested by incubation of Ne-15 with 1 per cent (w/v) sugar in 1 per cent (w/v) peptone solution. Acid formation was checked by the colour change of bromothymol blue in the culture medium. Mucous compound formation was checked by the incubation of Ne-15 in trypticase soy broth containing 5 per cent (w/v) sucrose. Pigmentation of the colony was checked on trypticase soy broth with 2 per cent (w/v) agar. Clzemicals Radioactive compounds were purchased from Daiichi Pure Chemicals, Tokyo. D(-) lactate and L( +) lactate (lithium salt), yeast alcohol dehydrogenase, L( +) lactate dehydrogenase (LDH) from rabbit muscle, D( -) LDH from Lactobacillus leichmannii, fungal glucose oxidase, horseradish peroxidase, pigheart malate dehydrogenase, acetate kinase from Escherichia coli and coenzymes (NAD+, NADH, NADP’, NADPH, cytochrome c, FMN, FAD, TPP, CoA, ATP, ADP and AMP) were from Boehringer, Germany. All chemicals obtained commercially were of reagent grade. Standard incubation system For the study of the degradation of carbohydrate and organic acid, the following standard incubation system was used. Cell suspension (equivalent to 5 mg dry weight of organism) was made up to 1.8 ml with an incubation mixture which had a final concentration of 0.1 M-potassium phosphate buffer, pH 7.0, 5 mM-MgCl, and 10 mM of each substrate. The mixtures were incubated at 35°C in a Warburg’s apparatus with shaking and the reaction was stopped by the addition of 0.2 ml of 50 per cent (w/v) perchloric acid solution. For one control, incubation was stopped before the addition of the substrate, and in another control the substrate was omitted. The CO,
678
Etsuro Hoshino. T. Yamada and S. Araya
evolved was absorbed in 0.2 ml of 20 per cent (w/v) KOH in the centre well of the vessel. In order to establish the presence of lactate degradation, ‘%-labelled substrate was added in some instances. In those experiments designed to detect CO2 fixation, labelled NaHi4C0, was added to the incubation mixture. Oxygen-uptake was measured during the incubation period. After incubation and centrifugation, the supernatant was subjected to chemical, enzymic and gas chromatographic analysis. In those experiments in which the substrate had been labelled with i4C, the radioactivity of the pellet and supernatant was determined. Analysis of’ organic acids Lactate was determined by the method of Barker and Summerson (1941). Isomers of lactate were determined enzymically, L(+) lactate as described by Hohorst (1965) and D(-) lactate as described by Gawehn and Bergmeyer (1974).Glucose was analyzed by the glucose oxidase method of Bergmeyer and Bemt (1965). Pyruvate was determined with lactate dehydrogenase (EC 1.1.1.27) (Biicher er al., 1965) and ethanol was measured with alcohol dehydrogenase (EC 1.1.1.1) (Bonnichsen, 1965). Acetate was estimated from acetohydroxamic acid formation with acetate kinase (EC 2.7.2.1) (Rose, 1955). Formate was separated by liquid chromatography using the technique of Belasco (1954) and the incorporated radioactivity from i4C-labelled substrate was counted. Radioactivity was measured by a liquid scintillation counter with 1Oml of scintillation fluid containing Triton X-100 which was prepared by the method of Patterson and Greene (1965). Keto acids were determined by the modified method described by Yamada and Carlsson (1973) and the incorporated radioactivity was counted. Gas chromatograph) Gas chromatography was as described by Carlsson and Griffith (1974), but 0.2 ml of acidified samples and control samples with 10N H,PO., were injected respectively into the column of the gas chromatography system (Packard, U.S.A.) without a pre-column of cation exchange resin. Methyl-esterized samples and control samples with diazomethane were also applied to the column. Analysis of the radioactil:ity incorporated into the centrijiqed pellet and the ninhydrin-positice fvaction The incubation mixture was centrifuged for 10min at 10,CNJOgat 4°C. After being washed and centrifuged, twice with 0.05 M-H2S04 and once with water, the pellet was suspended in scintillation fluid. The ninhydrin-positive fraction was separated from the supernatant with a cation-exchange resin column as described by Moffat and Lytle (1959) and the radioactivity was measured. Assn~ of‘02 uptake and CO, el;olution Oxygen uptake was determined manometrically. Evolved i4C02 from “C-labelled substrate was converted to Bai4C0, by the procedure described by Tsuiki and Kikuchi (1962) and suspended in scintillation fluid.
Assay of 14C0, ,jxation Twenty kLrnol of NaH14C0, was added to the lactate or pyruvate degradation system. KOH was omitted from the centre well. After the addition to perchloric acid, non-labelled CO, was passed through the incubation mixture for IOmin to eliminate non-reactive NaHi4C0,. After neutralization with K&O,, the radioactivity was measured. ASSUJ. of enzymes Nicotinamide adenine dinucleotide (NAD)-independent L(+ ) and D( - ) lactate dehydrogenase (LDH, EC 1.1.2.3 and 1.1.2.4 respectively) activity was assayed by the decrease in extinction at 600nm of oxidized 2,6-dichlorophenolindiphenol (DCPIP) in 3 ml of 0.1 M-potassium phosphate buffer, pH 7.0, containing 10,nmol of MgCl*, 0.1 lcmol of DCPIP, 10 pmol of L( +) lactate or D( -) lactate (lithium salt) and enzyme (2.5 mg as crude protein). When lactate or enzyme was omitted from the complete reaction system, no decrease in extinction at 600nm was detected. 0.16,nmol of cytochrome c was substituted for DCPIP and the activity was assayed by the increase in extinction at 550 nm. Other enzymes were assayed as follows: NADdependent LDH (EC 1.1.1.27 and 1.1.1.28) Stolzenbath (1966) and Stevenson and Holdsworth (1973); acetate kinase (EC 2.7.2.1) Rose (1955); acetyl-CoA synthetase (EC 6.2.1.1) Jones and Lipmann (1955); phosphate acetyltransferase (EC 2.3.1.8) Stadtman (1955); pyruvate dehydrogenase (EC 1.2.4.1) and oxoglutarate dehydrogenase (EC 1.2.4.2) Reed and Mukherjee (1969); isocitrate dehydrogenase (EC 1.1.1.42) Cleland, Thompson and Barden (1969); malate dehydrogenase (EC 1.1.1.37) Yoshida (1969); glutamate dehydrogenase (EC 1.4.1.3) Doherty (1970); malic enzyme (EC 1.1.1.40) Hsu and Lardy (1969); aconitate hydratase (EC 4.2.1.3) Fransler and Lowenstein (1969); fumarate hydratase (EC 4.2.1.2) Hill and Bradshaw (1969); aspartase (EC 4.3.1.1) Williams and Lartigue (1969): succinate dehydrogenase (EC 1.3.99.1) Veeger, Der Vartanian and Zeylemaker (1969); pyruvate oxidase (EC 1.2.3.3) Moyed and O’Kane (1956). Succinyl-CoA synthetase (EC 6.2.1.5) was assayed by measuring succinyl hydroxamic acid formation in the system as described by Bridger, Ramaley and Boyer (1969). Lactate racemase (EC 5.1.2.1) activity was assayed by the isometric conversion of lactate in 0.1 M-potassium phosphate buffer, pH 7.0. The converted isomer was detected enzymitally. RESULTS
Some biological characteristics
of’Neisseria
Neisseria strain Ne-15 was confirmed to be a Gram-negative diplococcus. This Neisseria grew well on ordinary culture media without blood or blood serum and fermented glucose, fructose, maltose or sucrose. Bright yellow chromogenesis was observed. Catalase and oxidase tests were positive. Mucous compound was formed from sucrose. Lactate degradation by intact cells of Ne-15 The optimum pH of lactate degradation by resting cells of Ne-15 under aerobic condition was 7.0. At
619
Lactate degradation by an oral Neisseria Table 1. Lactate ~mol/~mol
degradation
by Neisseria
of lactate consumption/30 mean * 1.05 0.91 0.80
O,-uptake CO, evolution Acetate formation
2. Detection
min SD (n = 4) * 0.11 * 0.02 * 0.14
Pathwuys from lactutr to acetute via pyruvute
pH 4.0 in 0.1 M-citrate buffer, lactate degradation was 16 per cent of that at pH 7.0. When 1 mol of lactate was consumed, about 1 mol of O+ptake and CO2 evolution was observed. Approximately 80 per cent of degraded lactate was converted to acetate (Table 1). A small amount of pyruvate and 2-oxoglutarate was detected on a thin-layer chromatogram for keto acid analysis (Table 2). However, acetoin, ethanol, methanol, propiocic acid and formic acid were not detected by gas chromatography. The absence of ethanol and formate formation was confirmed by enzyme analysis and liquid chromatography respectively. Table
Ne-15
When the Neisseriu resting cells were incubated with [1-r4C]-~(+) or D(-) lactate, the radioactivity was incorporated into pyruvate, suggesting the presence of pathways from lactate to pyruvate (Table 2). The activities of NAD-independent-L( +) and D( -) lactate dehydrogenase (EC 1.1.2.3 and 1.1.2.4 respectively) associated with these pathways were found in the cell-free extract. Neither NAD+ nor NADP+ stimulated the reduction of DCPIP (Table 3). Cytochrome c could substitute for DCPIP as an effective hydrogen acceptor.
of [14C]-labelled keto acids after incubation various substrates for 15 min
of Neisseriu
Radioactivity Substrate* Exp. 1 [l-‘“Cl D( -) Lactate [l-‘“Cl L( +) Lactate Exp. 2. [U-‘“Cl D( -) Lactate Exp. 3. [l-i4C] D( -) Lactate [U-‘“Cl Pyruvate * Specific activity: Table
3. Assay
[l-‘“Cl
of lactate
into
2-oxoglutarate
dpm/ml 1005 1155
dpm/ml 2285 2000
4.52
330
6755
2.78 4.52
843
2195 6030
0.0074 mCi/mmol,
dehydrydrogenase Neisseria
Exp. 1. Substrates
activity
others 0.01 mCi/mmol. in the
cell-free
extract
Ne- 15
BE at 600 nm/min/mg
protein
0.00 0.078 0.078 0.078 0.72 0.12 0.72
None L( + ) Lactate L(S) Lactate + NAD+ L(+) Lactate + NADP+ D( -) Lactate D(-) Lactate + NAD+ D( - ) Lactate + NADP+
pmol of NADH oxidized or NAD+ reduced/min/mg protein
Exp. 2. Substrates
0.007* O.GQOO 0.0000 0.0000 0.0000
Pyruiate + NADH L(+) Lactate + NAD+ L(+) Lactate + NADP’ D(-) Lactate + NAD’ D(-) Lactate + NADP+ * Data were compensated
incorporated
pyruvate pmol/ml 4.32 4.81
D( -)-lactate
Ne-15
for NADH
oxidase.
of
with
680
Etsuro Hoshino, T. Yamada and S. Araya Table 4. Pyruvate
degradation
by the Neisseria Ne-15 ~mol/~mol of pyruvate consumption/30 min mean f SD (n = 3) 0.14 + 0.06 0.68 f 0.06 1.28 _t 0.03
Lactate formation Acetate formation CO, evolution
Moreover, the cell-free extract of Ne-15 had marked peaks of absorption at 415, 520 and 550 nm. An addition of reductant (Na,S202) made these peaks more distinct. These peaks corresponded with that of ferrocytochrome c (Lenhoff and Kaplan, 1955). The activities of NAD-dependent LDH (EC 1.1.1.27 and 1.1.1.28) were also detected in the cell-free extract, but only the NADH-dependent reduction of pyruvate to L(+) or D(-) lactate were observed. Oxidation of L( +) or D(-) lactate by NADf or NADP+ was not found (Table 3). Lactate racemase activity was not detected. Ne-15 also degraded pyruvate to acetate and CO2 (Table 4). The activities of enzymes associated with the pathways from pyruvate to acetate, pyruvate dehydrogenase, acetyl-CoA synthetase, acetate kinase Table 5. Dilution
and phosphate acetyltransferase were detected in the cell-free extract, but pyruvate oxidase activity (Moyed and O’Kane, 1956) was not detected. An alternative pathway from lactate to acetate If the pathway through pyruvate had been the only catabolic pathway of lactate in Ne-15, the addition of unlabelled pyruvate to the lactate degradation system would have reduced the incorporation of radioactivity into CO2 from [14C]-lactate. However, the addition of unlabelled pyruvate did not reduce the r4C02 formation much (Table 5). A similar result was obtained during incubation with the cell-free extract. The direct pathway from lactate to acetate, not via pyruvate, was also expected from the experimental
effect of pyruvate
for 14C0,
evolution
Radioactivity
Substratest Exp. 1. (15 min incubation) [l-14C]-DL Lactate [1-r4C]-D~ Lactate + pyruvate* Exp. 2. (30 min incubation) [l-‘“Cl-t@ -) Lactate [l-‘“Cl-@ -) Lactate + pyruvate* [l-r4C]-L( +) Lactate [l-14C]-L( +) Lactate + pyruvate*
degradation
34,828 23,294 42,469 29,722
consumption
extract of Neisseria Ne-15 in the system incubated for 30 min -)
~mol/ml 2.90
1.54 1.11 1.41
Exp. 2. Gase phase [U-‘“Cl Pyruvate COz evolution Acetate formation
others 0.01 mCi/mmol.
LDH-M-R(
Exp. 1.
[1-r4C]-~~ Lactate Pyruvate formation CO2 evolution Acetate formation
by cell-free
consumptiont
into CO,
dpm 14,942 9828
* 20pmol were added to the incubation mixture. t Specific activity: [I -14C]-~( -)-lactate 0.0074 mCi/mmol, Table 6. Lactate
incorporated
LDH-M-R( +)* Nz gas pmol/ml 0.50 0.00 0.00
containing
LDH-M-R*
LDH-M-R(
+)*
pmol/ml 0.44 0.00 0.42 0.58 LDH-M-R( air
+)*
pmol/ml 0.50 0.23 0.24
* 180 units of LDH-M-R (LDH from rabbit muscle) and 10 pmol of NADH were added to incubation mixture. tPyruvate for substrate was reduced to 1 pmol (0.5 mM) and all the pyruvate was converted to lactate by LDH-M-R. Specific activity: [1-r4C]-~~ lactate, [U-r4C]-pyruvate 0.01 mCi/mmol
Lactate degradation by an oral Neisseria Table 7. O,-uptake @mol/60 min) by Neisseria Ne-15 after addition elf citric acid cycle intermediates 0.60 7.01 7.01 7.82 9.46 0.41 0.32 0.37 1.56 9.70 7.67
Citrate 2-Oxoglutarate Succinate Fumarate Malate Asparate Glutamate Acetate Glucose Pyruvate Lactate
data (Table 6). When sufficient LDH from rabbit muscle was added to the lactate degradation system of the cell-free extract, some lactate was consumed, pyruvate was not produced and stoichiometrical acetate formation and CO1 evolution from lactate were observed (Table 6, Exp. 1). Furthermore, r4C02 evolution from [“‘C]-pyruvate was not detected under anaerobic condition (Table 6, Exp. 2). These results suggest that a part of the lactate was converted directly to acetate by lactate oxidase (EC 1.1.3.2). Table 8. Inhibition
The presence of the citric acid cycle
As shown in Table 7, 2-oxoglutarate, succinate, fumarate and malate were utilized as substrate for O,-uptake by resting cells. For acetate degradation, it was essential to add a precursor of oxaloacetate, for example, succinate. However this degradation process, as demonstrated by 14C02 evolution from [14C]-acetate, was inhibited by the addition of monofluoroacetate (Table 8). Where resting cells were incubated with [‘4C]-lactate or [14C]-pyruvate, labelled 2-oxoglutarate was detected (Table 2). Additionally the enzyme activities associated with the citric acid cycle (aconitate hydratase, isocitrate dehydrogenase, succinyl-CoA synthetase, fumarate hydratase and malate dehydrogenase) were found in the cell-free extract. These results suggest that some lactate was degraded via the citric acid cycle. The 14C02 evolved from [2,3-*4C]-succinate by the resting cells was one tenth of that from [1,4-t4C]succinate. When 22,200 dpm of [U-U-‘4C]-lactate was con198 + 29dpm sumed by the resting cells, (mean + S.D., n = 4) of radioactivity were mcorporated into a ninhydrin-positive fraction and this was increased to 523 dpm by the addition of 10 pmol of NH,Cl to the incubation mixture. After centrifugation, the radioactivity from [U-‘4C]-lactate in the sediment increased with time as follows; 920dpm
of acetate metabolism by monofluoroacetate
(FA)
Radioactivity incorporated into CO,/90 min
Substrate
[‘“Cl [‘“Cl [“Cl [‘“Cl
681
dpm/2 ml 72 0 2790 0
Acetate Acetate + FA* Acetate + succinatet Acetate + FA* + succinatet
* Some cells (the same dry weight) were treated with monofluoroacetate (FA) before the incubation. 5 pmol of FA and 0.5 pmol of succinate per 1 mg dry weight of cells were added to the cell suspension for 60 min to make monofluorocitrate at room temperature. The cells used for other incubation were also left at room temperature for 60min. t 10 nmol. Spf:cific activity: [r4C]-acetate 0.01 mCi/mmol. Table 9. r4C02 fixation from NaHi4COJ in the cell-free extract of Neisseria Ne-15 Radioactivity fixed from NaH’4C0,/60
Complete system - Pyruvate - ATP - Enzyme - Acetyl-CoA - ATP + NADPH*
min
dpm 8729 959 0 0 4311 0
The cell-free incubation medium contained 10pmol of ATP, 10pmol of MgCl,, 10pmol of potassium pyruvate, 20pmol of NaH14C0,, 0.1 mg of acetyl-CoA and enzyme (7 mg as crude protein) in 2 ml of 0.1 M-potassium phosphate buffer, pH 7.0. Specific activity: NaH14C0, 0.0062 mCi/mmol. * NADPH substituted for ATP
682
Etsuro Hoshino, T. Yamada and S. Araya Table
10. Mixed incubation
Microorganisms
Glucose
consumption
~mol/ml/l5 3.20 0.05 3.10
PK 1 Ne-15 PK 1 + Ne-15
Ne-15 with Streptococcus
of resting cells of Neissrria mutans PK 1
Lactate formation
min
pmol/ml/l5 5.45 0.00 0.17
min
* For mixed incubation the system was not standard, but contained 15 prnol of MgCl,, 20pmol of glucose and 5 mg dry weight each cell suspension of PK 1 and Ne-15 in 3 ml of 0.1 M-potassium phosphate buffer, pH 7.0 (15min), 2251 dpm (30min), 4103 dpm (45min). By the addition of 1Opmol of NH&l to the incubation mixture, an increase of 1.75 times was observed at 30 min. The activities of glutamate dehydrogenase, aspartase and malic enzyme were also detected in the cell-free extract. CO2 jixation
NADH
during lactate degradution Acetyl -CoA*eb AMP ATP
When NaH14C0, was added to an incubation mixture of resting cells with lactate or pyruvate, radioactivity was detected from the incubation mixture after removing the unincorporated NaH14C0,. Pyruvate was an effective bicarbonate acceptor in the system of the cell-free extract. ATP was essential for, and acetyl-CoA stimulated, CO1 fixation. These results suggest the presence of pyruvate carboxylase (EC 6.4.1.1) in Ne-15. NADPH could not substitute for ATP in this system of CO* fixation, so malic enzyme apparently did not catalyze CO, fixation (Table 9). Mixed mutans
NAD+
incubation
of Neisseria
with
Oxoloacetote
\
\
DISCUSSION
Ne-15 degraded both L( +) lactate and D( - ) lactate under aerobic conditions. About 80 per cent of degraded lactate was converted to acetate and CO,. Most of the lactate was degraded via pyruvate to acetate and a part of lactate was converted to acetate directly by lactate oxidase. A small part of pyruvate was further metabolized via the citric acid cycle after CO2 fixation or after conversion to acetyl-CoA. Acetate was also metabolized via the citric acid cycle when oxaloacetate was present. The summarized pathways of lactate degradation by Ne-15 are shown in Fig. 1. The ability to convert lactate to pyruvate by NADindependent L( + ) and D( -) LDH is distributed widely among microorganisms (Stevenson and Holdsworth, 1973); NADdependent LDH reactions did not occur with Ne-15. The reduction of cytochrome c by lactate was catalyzed by NAD-independent LDH. As
cltrtc
Fumarate IV Asparate
Streptococcus
As shown in Table 10, Strep. mutarzs PK 1 produced lactate from glucose. The addition of Neisseria to the incubation mixture did not influence the quantity of glucose consumption but diminished lactate formation. This result suggests that Ne-15 degrades lactate which was produced by Strep. mutans PK 1 under aerobic conditions.
tialate \
Fig.
1. Proposed
Acetote
Cttrate
acid cycle
/ 2-oxoglutorate
pathways of Ne-15.
Gl:tamote
lactate
degradation
by
Neisseria have cytochrome oxidase (Berger, 1963), electrons are transferred to oxygen and oxidative phosphorylation may occur. Moreover, ATP might be generated during the reaction from acetyl-CoA to acetate (Jones and Lipmann, 1955; Rose, 1955). Although Ne-15 used the citric acid cycle, only a small portion of lactate was metabolized by that pathway. The citric acid cycle may be considered as a synthetic, rather than a catabolic, pathway. As 14C02 formation from [2,3-14C]-succinate was small compared with that from [1,4-14C]-succinate, many intermediates of this cycle may have been removed for synthetic reactions. An increase in the synthesis of cell components by the addition of ammonia to the incubation mixture supports this hypothesis. Lactate produced by Strep. mutans was degraded mainly to acetate by Ne-15. This conversion of lactate to a weak and volatile acid suggests the possibility that the presence of Neisseria in dental plaque may reduce the initiation and progress of dental caries similarly to that by Veillonella in experimental animals (Mikx et al., 1972). Ackmwledymenr-This investigation was supported in part by The Mitsubishi Foundation. We wish to thank Dr. G. H. Bowden for his kind suggestions and confimation about characteristics of Ne-15.
683
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Bergmeyer H.-U. anti Bernt E. 1965. Methods in Enzymatic Analysis (Edited by Bergmeyer H.-U. and translated by Williamson D. H.), pp. 123-128. Academic Press, New York. Bonnichsen R. 1965. Methods in Enzymatic Analysis. (Edited by Bergmeyer H.-U. and translated by Williamson D. H.). DD. :!85%287.Academic Press. New York. Bridger W. A., Ramaley R. F. and Boyer P. D. 1969. Methods in Enzymology, Vol. 13 (Edited by Lowenstein J. M.), p. 70. Academic Press, New York. Biicher T., Czok R., Lamprecht W. and Latzko E. 1965. Methods in Enzymatic Analysis (Edited by Bergmeyer H.-U. and translated by Williamson D. H.), pp. 253259. Academic Press, New York. Carlsson J. and Griffith C. J. 1974. Fermentation products and bacterial yields in glucose-limited and nitrogenlimited cultures of streptococci. Archs orul Biol. 19, 1105-~1109. Cleland W. W., Thompson V. W. and Barden R. E. 1969. Methods in Enzymology, Vol. 13 (Edited by Lowenstein J. M.), p. 30. Academic Press, New York. Doherty D. 1970. Methods in Enzymology, Vol. 17, A (Edited by Tabo,- H. and Tabor C. W.), pp. 850-852. Academic- Press, New York. Fransler B. and Lowenstein J. M. 1969. Methods in Enzvmoloqv, Vol. 13 (Edited by Lowenstein J. M.), pp. __ 26-25. Academic Press, New Y&k. Cawehn K. and Bergmever H.-U. 1974. Methods in Enxmatic Analysis, 2ndYEd: (Edited by Bergmeyer H.-U. aid translated by W lliamson D. H.). pp. 1492-1495. Academic Press, New York. Geddes D. A. M. 1972. The production of L(+) and II-) lactic acid and volatile acids by human dental plaque and the effect of plaque buffering and acidic strength on DH. Archs oral Biol. 17. 537-545. Gedd& D. A. M. 1975. Acids produced by human dental plaque metabolism in situ. Caries Rex 4, 98-109. Gibbons R. J.. Berman K. S., Knoettner R. and Kaosimalis B. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule-forming streptococci of human origin. Avhs oral Biol. 11, 549-560. Gibbons R. J. and Nygaard M. 1968. Synthesis of insoluble dextran and its significance in the formation of gelatinous deposits b; plaque-forming streptococci. Archs oral Biol. 13, 12‘19%1262. Hemmens E. S., B:ayney J. R., Bradel S. F. and Harison R. W. 1946. Th: microbic flora of the dental plaque in relation to the beginning of caries. J. dent. Res. 25, 195-205. Hill R. L. and Bradshaw R. A. 1969. Methods in Enzymology, Vol. 13 (Edited by Lowenstein J. M.), pp. 91-92. Academic Press, New York. Hohorst H.-J. 1965. Methods in Enzymatic Anulysis (Edited by Bergmeyer H.-U. and translated by Williamson D. H.), pp. 266270. Academic Press, New York. Hsu R. Y. and Lardy H. A. 1969. Methods in Enzymology, Vol. 13 (Edited by Lowenstein J. M.), pp. 230-231. Academic Press, New York.
Jones N. E. and Lipmann F. 1955. Methods in Enzymology, Vol. 1 (Edited by Colowich S. P. and Kaplan N. O.), pp. 585-587. Academic Press, New York. Kovacs N. 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature (Land.) 178, 703. Lenhoff H. M. and Kaplan N. 0. 1955. Methods in Enzymology, Vol. 2 (Edited by Colowick S. P. and Kaplan N. O.), p. 758. Academic Press, New York. Mikx F. H. M., Hoeven J. S. van der, KGnig K. G., Plasschaert A. J. M. and Guggenheim B. 1972. Establishment of defined microbial ecosystems in germ-free rat. 1. The effect of the interaction oiStreptoco&s mutuns or Streprococcus sanguis with I/eillonella alcalescenfs on plaque formation and caries activity. Curies Res. 6, 211-223. Moffat E. D. and Lytle R. I. 1959. Polychromatic technique for the identification of amino acids on paper chromatogram. Analvt. Chem. 31, 92&928. Morris E. 0: 1954. The bacteriology of the oral cavity. IV (B) Neisseria. Br. dent. J. 96, 263-264. Moyed H. S. and O’Kane D. J. 1956. Enzymes and coenzymes of the pyruvate oxidase of Proreus. J. biol. Chem. 218, 831-840. Muntz J. A. 1943. Production of acids from glucose by dental plaque material. J. biol. Chem. 148, 225-236. Patterson M. S. and Greene R. C. 1965. Measurement of low energy beta-emitters in aquenous solution by liquid scintillation counting of emulsions. Analyt. Chem. 37, 854859.
Reed L. J. and Mukherjee B. B. 1969. Methods in Enzymoloav. Vol. 13 (Edited bv Lowenstein J. M.). ,, DD. II 5&57. Academic Press, New ?ork. Ritz H. L. 1967. Microbial population shifts in developing human dental plaque. Archs oral Biol. 12, 1561-1568. Ritz H. L. 1969. Fluorescent antibody staining of Neisseria, Srreptococcus and Veillonella in frozen sections of human dental plaque. Archs orul Biol. 14, 1073-1083. Rose I. A. 1955. Methods in Enzymology, Vol. 1 (Edited by Colowick S. P. and Kaplan N. O.), p. 592. Academic Press, New York. Sidaway D. A. 1969. The bacterial composition of natural plaque and the in vitro production of artificial plaque. In: Dental Plaque (Edited by McHugh W. D.), pn. .. 225-240. Thompson, Dundee. _ Stadtman E. R. 1955. Methods in Enzymology, Vol. 1 (Edited by Colowick S. P. and Kaplan N. O.), pp. 596-597. Academic Press, New York. Stevenson P. M. and Holdsworth E. S. 1973. The distribution of NAD-independent lactic dehydroeenases amongst microorganiims. J. gen. Microbial 78,-83-88. Stolzenbach F. 1966. Methods in Enzvmoloav. Vol. 9 (Edited by Wood W. A.), pp. 278-279.‘Acad&ic Press. New York. Tsuiki S. and Kikuchi G. 1962. Catabolism of glycine in .I,
Rhodopseudomonas
sheriodes.
Biochem.
biophps. Acta 64,
514-525. Veeger C., Vartanian D. V. and Zeylemaker W. P. 1969. Methods in Enzvmoloav. Vol. 13 (Edited bv Lowenstein J. M.), p. 83. Academic’Press, New York. a Williams V. R. and Lartigue D. J. 1969. Methods in Enzymology, Vol. 13 (Edited by Lowenstein J. M.), pp. 354-355. Academic Press, New York. Yamada T. and Araya S. 1975. The role of transamination in ammonium assimilation in Streptococcus sanguis. Archs oral Biol. 20. 44549. Yamada T. and Carlsson J. 1973. Phosphoenolpyruvate carboxylase and ammonium metabolism in oral streptocci. Ar_chs oral Biol. 18, 799-812. Yoshida A. 1969. Methods in Enzymology, Vol. 13 (Edited by Lowenstein J. M.), p. 141. Academic Press, New York.
Vol. ?I. up. 677 lo 683. Pergamon
Press 1976 Prmted m Great Brltam
LACTATE DEGRADATION BY A STRAIN OF NEISXERIA ISOLATED FROM HUMAN DENTAL PLAQUE ETSURO HOSHINO, T. YAMADA and S. ARAYA Laboratory of Oral Biochemistry, Tohoku University, School of Dentistry, Sendai, 980 Japan Summary--An oral Neisseriu strain Ne-15 degraded lactate under aerobic conditions. Most of the lacr ate, both the L( + ) and D( - ) forms, was converted via pyruvate to acetate, and it seems that ATP may be generated through this pathway. Part of the lactate was converted to acetate directly. A small portion of pyruvate was further metabolized by the way of the citric acid cycle after CO, fixation or after conversion to acetyl-CoA, and was utilized for synthesis cf cell components. The addition of the Neisseria in a mixed incubation with Streptococcus mu~ans caused no change in glucose utilization but a reduced lactate concentration.
INTRODUCTION Dental plaque de;;rades glucose not only to lactate but also to volatile acids and under aerobic conditions rapid destruction of lactic acid occurs at neutral pH (Muntz, 1943). Lactate may be converted to some weak and volatile acids which are predominant in ‘resting plaque’ irr ~iuo (Geddes, 1972; 1975). Veillonella, one of the microorganisms which degrade lactate and form volatile acids, reduces the cariogenic activity of Strrptococcus mutans in rats (Mikx et u1., 1972). The concomitant presence of Neisseria in denta plaque (Hemmens et al., 1946; Morris, 1954; Ritz, 1967 and 1969; Slidaway, 1969) and their ability to metabolize lactate (Berger, 1962) may imply that these microorganisms take a part in the degradation of lactate in dental plaque and play an important role in the microbial ecology of the plaque. MATERIALSAND
METHODS
M icrovrganisnzs Neisseria strair 15 (Ne-15) isolated from human dental plaque was obtained from Prof. I. Takazoe, Tokyo Dental College, and cultured in shallow layer (about 1Omm deep) of trypticase soy broth (BBL, Cockeysville) at 3 5°C with shaking. Each culture was Gram-stained, tested for the presence of oxidase (Kovacs, 1956) and for catalase. Ne-15 was identified as a saccharolytic Neisseria similar to that isolated from the dental plaque of children in England (G. H. Bowden, personal communication). Streptococcus mutans PK 1 (Gibbons et a/., 1966) was supplied by Prof. W. Kondo, School of Dentistry, Niigata University, Niigata, and was cultured in Gibbons and Nygaard’s (1968) medium supplemented with 2 per cent (w/v) glucose and 0.1 per cent (w/v) yeast extract. The purity was checked on blood agar. Both organisms were harvested during their logarithmic growth phase by centrifugation and washed three times with 40 mM-potassium phosphate buffer, pH 7.0, at 4°C and suspended in the same buffer. The dry weight of the cells was estimated from the turbidity of the suspension at 660nm.
The same cells of Ne-15 were subjected to enzyme assay. A cell-free extract of Ne-15 was prepared by the method described by Yamada and Araya (1975). Characterization
I$ Ne-15
Fermentation of glucose, fructose, maltose or sucrose was tested by incubation of Ne-15 with 1 per cent (w/v) sugar in 1 per cent (w/v) peptone solution. Acid formation was checked by the colour change of bromothymol blue in the culture medium. Mucous compound formation was checked by the incubation of Ne-15 in trypticase soy broth containing 5 per cent (w/v) sucrose. Pigmentation of the colony was checked on trypticase soy broth with 2 per cent (w/v) agar. Clzemicals Radioactive compounds were purchased from Daiichi Pure Chemicals, Tokyo. D(-) lactate and L( +) lactate (lithium salt), yeast alcohol dehydrogenase, L( +) lactate dehydrogenase (LDH) from rabbit muscle, D( -) LDH from Lactobacillus leichmannii, fungal glucose oxidase, horseradish peroxidase, pigheart malate dehydrogenase, acetate kinase from Escherichia coli and coenzymes (NAD+, NADH, NADP’, NADPH, cytochrome c, FMN, FAD, TPP, CoA, ATP, ADP and AMP) were from Boehringer, Germany. All chemicals obtained commercially were of reagent grade. Standard incubation system For the study of the degradation of carbohydrate and organic acid, the following standard incubation system was used. Cell suspension (equivalent to 5 mg dry weight of organism) was made up to 1.8 ml with an incubation mixture which had a final concentration of 0.1 M-potassium phosphate buffer, pH 7.0, 5 mM-MgCl, and 10 mM of each substrate. The mixtures were incubated at 35°C in a Warburg’s apparatus with shaking and the reaction was stopped by the addition of 0.2 ml of 50 per cent (w/v) perchloric acid solution. For one control, incubation was stopped before the addition of the substrate, and in another control the substrate was omitted. The CO,
678
Etsuro Hoshino. T. Yamada and S. Araya
evolved was absorbed in 0.2 ml of 20 per cent (w/v) KOH in the centre well of the vessel. In order to establish the presence of lactate degradation, ‘%-labelled substrate was added in some instances. In those experiments designed to detect CO2 fixation, labelled NaHi4C0, was added to the incubation mixture. Oxygen-uptake was measured during the incubation period. After incubation and centrifugation, the supernatant was subjected to chemical, enzymic and gas chromatographic analysis. In those experiments in which the substrate had been labelled with i4C, the radioactivity of the pellet and supernatant was determined. Analysis of’ organic acids Lactate was determined by the method of Barker and Summerson (1941). Isomers of lactate were determined enzymically, L(+) lactate as described by Hohorst (1965) and D(-) lactate as described by Gawehn and Bergmeyer (1974).Glucose was analyzed by the glucose oxidase method of Bergmeyer and Bemt (1965). Pyruvate was determined with lactate dehydrogenase (EC 1.1.1.27) (Biicher er al., 1965) and ethanol was measured with alcohol dehydrogenase (EC 1.1.1.1) (Bonnichsen, 1965). Acetate was estimated from acetohydroxamic acid formation with acetate kinase (EC 2.7.2.1) (Rose, 1955). Formate was separated by liquid chromatography using the technique of Belasco (1954) and the incorporated radioactivity from i4C-labelled substrate was counted. Radioactivity was measured by a liquid scintillation counter with 1Oml of scintillation fluid containing Triton X-100 which was prepared by the method of Patterson and Greene (1965). Keto acids were determined by the modified method described by Yamada and Carlsson (1973) and the incorporated radioactivity was counted. Gas chromatograph) Gas chromatography was as described by Carlsson and Griffith (1974), but 0.2 ml of acidified samples and control samples with 10N H,PO., were injected respectively into the column of the gas chromatography system (Packard, U.S.A.) without a pre-column of cation exchange resin. Methyl-esterized samples and control samples with diazomethane were also applied to the column. Analysis of the radioactil:ity incorporated into the centrijiqed pellet and the ninhydrin-positice fvaction The incubation mixture was centrifuged for 10min at 10,CNJOgat 4°C. After being washed and centrifuged, twice with 0.05 M-H2S04 and once with water, the pellet was suspended in scintillation fluid. The ninhydrin-positive fraction was separated from the supernatant with a cation-exchange resin column as described by Moffat and Lytle (1959) and the radioactivity was measured. Assn~ of‘02 uptake and CO, el;olution Oxygen uptake was determined manometrically. Evolved i4C02 from “C-labelled substrate was converted to Bai4C0, by the procedure described by Tsuiki and Kikuchi (1962) and suspended in scintillation fluid.
Assay of 14C0, ,jxation Twenty kLrnol of NaH14C0, was added to the lactate or pyruvate degradation system. KOH was omitted from the centre well. After the addition to perchloric acid, non-labelled CO, was passed through the incubation mixture for IOmin to eliminate non-reactive NaHi4C0,. After neutralization with K&O,, the radioactivity was measured. ASSUJ. of enzymes Nicotinamide adenine dinucleotide (NAD)-independent L(+ ) and D( - ) lactate dehydrogenase (LDH, EC 1.1.2.3 and 1.1.2.4 respectively) activity was assayed by the decrease in extinction at 600nm of oxidized 2,6-dichlorophenolindiphenol (DCPIP) in 3 ml of 0.1 M-potassium phosphate buffer, pH 7.0, containing 10,nmol of MgCl*, 0.1 lcmol of DCPIP, 10 pmol of L( +) lactate or D( -) lactate (lithium salt) and enzyme (2.5 mg as crude protein). When lactate or enzyme was omitted from the complete reaction system, no decrease in extinction at 600nm was detected. 0.16,nmol of cytochrome c was substituted for DCPIP and the activity was assayed by the increase in extinction at 550 nm. Other enzymes were assayed as follows: NADdependent LDH (EC 1.1.1.27 and 1.1.1.28) Stolzenbath (1966) and Stevenson and Holdsworth (1973); acetate kinase (EC 2.7.2.1) Rose (1955); acetyl-CoA synthetase (EC 6.2.1.1) Jones and Lipmann (1955); phosphate acetyltransferase (EC 2.3.1.8) Stadtman (1955); pyruvate dehydrogenase (EC 1.2.4.1) and oxoglutarate dehydrogenase (EC 1.2.4.2) Reed and Mukherjee (1969); isocitrate dehydrogenase (EC 1.1.1.42) Cleland, Thompson and Barden (1969); malate dehydrogenase (EC 1.1.1.37) Yoshida (1969); glutamate dehydrogenase (EC 1.4.1.3) Doherty (1970); malic enzyme (EC 1.1.1.40) Hsu and Lardy (1969); aconitate hydratase (EC 4.2.1.3) Fransler and Lowenstein (1969); fumarate hydratase (EC 4.2.1.2) Hill and Bradshaw (1969); aspartase (EC 4.3.1.1) Williams and Lartigue (1969): succinate dehydrogenase (EC 1.3.99.1) Veeger, Der Vartanian and Zeylemaker (1969); pyruvate oxidase (EC 1.2.3.3) Moyed and O’Kane (1956). Succinyl-CoA synthetase (EC 6.2.1.5) was assayed by measuring succinyl hydroxamic acid formation in the system as described by Bridger, Ramaley and Boyer (1969). Lactate racemase (EC 5.1.2.1) activity was assayed by the isometric conversion of lactate in 0.1 M-potassium phosphate buffer, pH 7.0. The converted isomer was detected enzymitally. RESULTS
Some biological characteristics
of’Neisseria
Neisseria strain Ne-15 was confirmed to be a Gram-negative diplococcus. This Neisseria grew well on ordinary culture media without blood or blood serum and fermented glucose, fructose, maltose or sucrose. Bright yellow chromogenesis was observed. Catalase and oxidase tests were positive. Mucous compound was formed from sucrose. Lactate degradation by intact cells of Ne-15 The optimum pH of lactate degradation by resting cells of Ne-15 under aerobic condition was 7.0. At
619
Lactate degradation by an oral Neisseria Table 1. Lactate ~mol/~mol
degradation
by Neisseria
of lactate consumption/30 mean * 1.05 0.91 0.80
O,-uptake CO, evolution Acetate formation
2. Detection
min SD (n = 4) * 0.11 * 0.02 * 0.14
Pathwuys from lactutr to acetute via pyruvute
pH 4.0 in 0.1 M-citrate buffer, lactate degradation was 16 per cent of that at pH 7.0. When 1 mol of lactate was consumed, about 1 mol of O+ptake and CO2 evolution was observed. Approximately 80 per cent of degraded lactate was converted to acetate (Table 1). A small amount of pyruvate and 2-oxoglutarate was detected on a thin-layer chromatogram for keto acid analysis (Table 2). However, acetoin, ethanol, methanol, propiocic acid and formic acid were not detected by gas chromatography. The absence of ethanol and formate formation was confirmed by enzyme analysis and liquid chromatography respectively. Table
Ne-15
When the Neisseriu resting cells were incubated with [1-r4C]-~(+) or D(-) lactate, the radioactivity was incorporated into pyruvate, suggesting the presence of pathways from lactate to pyruvate (Table 2). The activities of NAD-independent-L( +) and D( -) lactate dehydrogenase (EC 1.1.2.3 and 1.1.2.4 respectively) associated with these pathways were found in the cell-free extract. Neither NAD+ nor NADP+ stimulated the reduction of DCPIP (Table 3). Cytochrome c could substitute for DCPIP as an effective hydrogen acceptor.
of [14C]-labelled keto acids after incubation various substrates for 15 min
of Neisseriu
Radioactivity Substrate* Exp. 1 [l-‘“Cl D( -) Lactate [l-‘“Cl L( +) Lactate Exp. 2. [U-‘“Cl D( -) Lactate Exp. 3. [l-i4C] D( -) Lactate [U-‘“Cl Pyruvate * Specific activity: Table
3. Assay
[l-‘“Cl
of lactate
into
2-oxoglutarate
dpm/ml 1005 1155
dpm/ml 2285 2000
4.52
330
6755
2.78 4.52
843
2195 6030
0.0074 mCi/mmol,
dehydrydrogenase Neisseria
Exp. 1. Substrates
activity
others 0.01 mCi/mmol. in the
cell-free
extract
Ne- 15
BE at 600 nm/min/mg
protein
0.00 0.078 0.078 0.078 0.72 0.12 0.72
None L( + ) Lactate L(S) Lactate + NAD+ L(+) Lactate + NADP+ D( -) Lactate D(-) Lactate + NAD+ D( - ) Lactate + NADP+
pmol of NADH oxidized or NAD+ reduced/min/mg protein
Exp. 2. Substrates
0.007* O.GQOO 0.0000 0.0000 0.0000
Pyruiate + NADH L(+) Lactate + NAD+ L(+) Lactate + NADP’ D(-) Lactate + NAD’ D(-) Lactate + NADP+ * Data were compensated
incorporated
pyruvate pmol/ml 4.32 4.81
D( -)-lactate
Ne-15
for NADH
oxidase.
of
with
680
Etsuro Hoshino, T. Yamada and S. Araya Table 4. Pyruvate
degradation
by the Neisseria Ne-15 ~mol/~mol of pyruvate consumption/30 min mean f SD (n = 3) 0.14 + 0.06 0.68 f 0.06 1.28 _t 0.03
Lactate formation Acetate formation CO, evolution
Moreover, the cell-free extract of Ne-15 had marked peaks of absorption at 415, 520 and 550 nm. An addition of reductant (Na,S202) made these peaks more distinct. These peaks corresponded with that of ferrocytochrome c (Lenhoff and Kaplan, 1955). The activities of NAD-dependent LDH (EC 1.1.1.27 and 1.1.1.28) were also detected in the cell-free extract, but only the NADH-dependent reduction of pyruvate to L(+) or D(-) lactate were observed. Oxidation of L( +) or D(-) lactate by NADf or NADP+ was not found (Table 3). Lactate racemase activity was not detected. Ne-15 also degraded pyruvate to acetate and CO2 (Table 4). The activities of enzymes associated with the pathways from pyruvate to acetate, pyruvate dehydrogenase, acetyl-CoA synthetase, acetate kinase Table 5. Dilution
and phosphate acetyltransferase were detected in the cell-free extract, but pyruvate oxidase activity (Moyed and O’Kane, 1956) was not detected. An alternative pathway from lactate to acetate If the pathway through pyruvate had been the only catabolic pathway of lactate in Ne-15, the addition of unlabelled pyruvate to the lactate degradation system would have reduced the incorporation of radioactivity into CO2 from [14C]-lactate. However, the addition of unlabelled pyruvate did not reduce the r4C02 formation much (Table 5). A similar result was obtained during incubation with the cell-free extract. The direct pathway from lactate to acetate, not via pyruvate, was also expected from the experimental
effect of pyruvate
for 14C0,
evolution
Radioactivity
Substratest Exp. 1. (15 min incubation) [l-14C]-DL Lactate [1-r4C]-D~ Lactate + pyruvate* Exp. 2. (30 min incubation) [l-‘“Cl-t@ -) Lactate [l-‘“Cl-@ -) Lactate + pyruvate* [l-r4C]-L( +) Lactate [l-14C]-L( +) Lactate + pyruvate*
degradation
34,828 23,294 42,469 29,722
consumption
extract of Neisseria Ne-15 in the system incubated for 30 min -)
~mol/ml 2.90
1.54 1.11 1.41
Exp. 2. Gase phase [U-‘“Cl Pyruvate COz evolution Acetate formation
others 0.01 mCi/mmol.
LDH-M-R(
Exp. 1.
[1-r4C]-~~ Lactate Pyruvate formation CO2 evolution Acetate formation
by cell-free
consumptiont
into CO,
dpm 14,942 9828
* 20pmol were added to the incubation mixture. t Specific activity: [I -14C]-~( -)-lactate 0.0074 mCi/mmol, Table 6. Lactate
incorporated
LDH-M-R( +)* Nz gas pmol/ml 0.50 0.00 0.00
containing
LDH-M-R*
LDH-M-R(
+)*
pmol/ml 0.44 0.00 0.42 0.58 LDH-M-R( air
+)*
pmol/ml 0.50 0.23 0.24
* 180 units of LDH-M-R (LDH from rabbit muscle) and 10 pmol of NADH were added to incubation mixture. tPyruvate for substrate was reduced to 1 pmol (0.5 mM) and all the pyruvate was converted to lactate by LDH-M-R. Specific activity: [1-r4C]-~~ lactate, [U-r4C]-pyruvate 0.01 mCi/mmol
Lactate degradation by an oral Neisseria Table 7. O,-uptake @mol/60 min) by Neisseria Ne-15 after addition elf citric acid cycle intermediates 0.60 7.01 7.01 7.82 9.46 0.41 0.32 0.37 1.56 9.70 7.67
Citrate 2-Oxoglutarate Succinate Fumarate Malate Asparate Glutamate Acetate Glucose Pyruvate Lactate
data (Table 6). When sufficient LDH from rabbit muscle was added to the lactate degradation system of the cell-free extract, some lactate was consumed, pyruvate was not produced and stoichiometrical acetate formation and CO1 evolution from lactate were observed (Table 6, Exp. 1). Furthermore, r4C02 evolution from [“‘C]-pyruvate was not detected under anaerobic condition (Table 6, Exp. 2). These results suggest that a part of the lactate was converted directly to acetate by lactate oxidase (EC 1.1.3.2). Table 8. Inhibition
The presence of the citric acid cycle
As shown in Table 7, 2-oxoglutarate, succinate, fumarate and malate were utilized as substrate for O,-uptake by resting cells. For acetate degradation, it was essential to add a precursor of oxaloacetate, for example, succinate. However this degradation process, as demonstrated by 14C02 evolution from [14C]-acetate, was inhibited by the addition of monofluoroacetate (Table 8). Where resting cells were incubated with [‘4C]-lactate or [14C]-pyruvate, labelled 2-oxoglutarate was detected (Table 2). Additionally the enzyme activities associated with the citric acid cycle (aconitate hydratase, isocitrate dehydrogenase, succinyl-CoA synthetase, fumarate hydratase and malate dehydrogenase) were found in the cell-free extract. These results suggest that some lactate was degraded via the citric acid cycle. The 14C02 evolved from [2,3-*4C]-succinate by the resting cells was one tenth of that from [1,4-t4C]succinate. When 22,200 dpm of [U-U-‘4C]-lactate was con198 + 29dpm sumed by the resting cells, (mean + S.D., n = 4) of radioactivity were mcorporated into a ninhydrin-positive fraction and this was increased to 523 dpm by the addition of 10 pmol of NH,Cl to the incubation mixture. After centrifugation, the radioactivity from [U-‘4C]-lactate in the sediment increased with time as follows; 920dpm
of acetate metabolism by monofluoroacetate
(FA)
Radioactivity incorporated into CO,/90 min
Substrate
[‘“Cl [‘“Cl [“Cl [‘“Cl
681
dpm/2 ml 72 0 2790 0
Acetate Acetate + FA* Acetate + succinatet Acetate + FA* + succinatet
* Some cells (the same dry weight) were treated with monofluoroacetate (FA) before the incubation. 5 pmol of FA and 0.5 pmol of succinate per 1 mg dry weight of cells were added to the cell suspension for 60 min to make monofluorocitrate at room temperature. The cells used for other incubation were also left at room temperature for 60min. t 10 nmol. Spf:cific activity: [r4C]-acetate 0.01 mCi/mmol. Table 9. r4C02 fixation from NaHi4COJ in the cell-free extract of Neisseria Ne-15 Radioactivity fixed from NaH’4C0,/60
Complete system - Pyruvate - ATP - Enzyme - Acetyl-CoA - ATP + NADPH*
min
dpm 8729 959 0 0 4311 0
The cell-free incubation medium contained 10pmol of ATP, 10pmol of MgCl,, 10pmol of potassium pyruvate, 20pmol of NaH14C0,, 0.1 mg of acetyl-CoA and enzyme (7 mg as crude protein) in 2 ml of 0.1 M-potassium phosphate buffer, pH 7.0. Specific activity: NaH14C0, 0.0062 mCi/mmol. * NADPH substituted for ATP
682
Etsuro Hoshino, T. Yamada and S. Araya Table
10. Mixed incubation
Microorganisms
Glucose
consumption
~mol/ml/l5 3.20 0.05 3.10
PK 1 Ne-15 PK 1 + Ne-15
Ne-15 with Streptococcus
of resting cells of Neissrria mutans PK 1
Lactate formation
min
pmol/ml/l5 5.45 0.00 0.17
min
* For mixed incubation the system was not standard, but contained 15 prnol of MgCl,, 20pmol of glucose and 5 mg dry weight each cell suspension of PK 1 and Ne-15 in 3 ml of 0.1 M-potassium phosphate buffer, pH 7.0 (15min), 2251 dpm (30min), 4103 dpm (45min). By the addition of 1Opmol of NH&l to the incubation mixture, an increase of 1.75 times was observed at 30 min. The activities of glutamate dehydrogenase, aspartase and malic enzyme were also detected in the cell-free extract. CO2 jixation
NADH
during lactate degradution Acetyl -CoA*eb AMP ATP
When NaH14C0, was added to an incubation mixture of resting cells with lactate or pyruvate, radioactivity was detected from the incubation mixture after removing the unincorporated NaH14C0,. Pyruvate was an effective bicarbonate acceptor in the system of the cell-free extract. ATP was essential for, and acetyl-CoA stimulated, CO1 fixation. These results suggest the presence of pyruvate carboxylase (EC 6.4.1.1) in Ne-15. NADPH could not substitute for ATP in this system of CO* fixation, so malic enzyme apparently did not catalyze CO, fixation (Table 9). Mixed mutans
NAD+
incubation
of Neisseria
with
Oxoloacetote
\
\
DISCUSSION
Ne-15 degraded both L( +) lactate and D( - ) lactate under aerobic conditions. About 80 per cent of degraded lactate was converted to acetate and CO,. Most of the lactate was degraded via pyruvate to acetate and a part of lactate was converted to acetate directly by lactate oxidase. A small part of pyruvate was further metabolized via the citric acid cycle after CO2 fixation or after conversion to acetyl-CoA. Acetate was also metabolized via the citric acid cycle when oxaloacetate was present. The summarized pathways of lactate degradation by Ne-15 are shown in Fig. 1. The ability to convert lactate to pyruvate by NADindependent L( + ) and D( -) LDH is distributed widely among microorganisms (Stevenson and Holdsworth, 1973); NADdependent LDH reactions did not occur with Ne-15. The reduction of cytochrome c by lactate was catalyzed by NAD-independent LDH. As
cltrtc
Fumarate IV Asparate
Streptococcus
As shown in Table 10, Strep. mutarzs PK 1 produced lactate from glucose. The addition of Neisseria to the incubation mixture did not influence the quantity of glucose consumption but diminished lactate formation. This result suggests that Ne-15 degrades lactate which was produced by Strep. mutans PK 1 under aerobic conditions.
tialate \
Fig.
1. Proposed
Acetote
Cttrate
acid cycle
/ 2-oxoglutorate
pathways of Ne-15.
Gl:tamote
lactate
degradation
by
Neisseria have cytochrome oxidase (Berger, 1963), electrons are transferred to oxygen and oxidative phosphorylation may occur. Moreover, ATP might be generated during the reaction from acetyl-CoA to acetate (Jones and Lipmann, 1955; Rose, 1955). Although Ne-15 used the citric acid cycle, only a small portion of lactate was metabolized by that pathway. The citric acid cycle may be considered as a synthetic, rather than a catabolic, pathway. As 14C02 formation from [2,3-14C]-succinate was small compared with that from [1,4-14C]-succinate, many intermediates of this cycle may have been removed for synthetic reactions. An increase in the synthesis of cell components by the addition of ammonia to the incubation mixture supports this hypothesis. Lactate produced by Strep. mutans was degraded mainly to acetate by Ne-15. This conversion of lactate to a weak and volatile acid suggests the possibility that the presence of Neisseria in dental plaque may reduce the initiation and progress of dental caries similarly to that by Veillonella in experimental animals (Mikx et al., 1972). Ackmwledymenr-This investigation was supported in part by The Mitsubishi Foundation. We wish to thank Dr. G. H. Bowden for his kind suggestions and confimation about characteristics of Ne-15.
683
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