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Journal of General Microbiology (1984), 130, 1787-1793. Printed in Great Britain
1787
The Acid End-products of Glucose Metabolism of Oral and Other
Haemophili
By J . E. T U Y A U , ' W. S I M S 1 * t A N D R . A. D . W I L L I A M S 2
Department of Microbiology and Preventive Dentistry, Royal Dental Hospital, Leicester Square,
London WC2H 7LJ, UK
Department of Biochemistry, London Hospital Medical College, Turner Street,
London El 2AD, UK
(Received 5 December I983 ;revised 28 February 1984)
The acids produced in broth culture by various species of oral haemophili and by stock strains of
capsulated and other haemophili were identified and measured by gas-liquid chromatography.
Succinic acid was the major acid end-product of all strains, with acetic acid also being regularly
produced but in smaller amounts. A stock strain, Haemophilus paraintftuenzae NCTC 4101,
produced less succinic acid than other strains of this species and produced much more oxalacetic
and pyruvic acid than all the other strains of haemophili. Strain NCTC 4101 possessed all the
enzymes of the tricarboxylic acid cycle, as previously reported, but in the other haemophili
examined only succinic dehydrogenase, fumarase and malate dehydrogenase could be detected.
No other enzymes of the tricarboxylic acid cycle were detected and isocitrate lyase, malate
synthase and pyruvate carboxylase were also absent. Phosphoenolpyruvate-carboxylase was
present in all strains. A partial tricarboxylic acid cycle and marked malate dehydrogenase
activity appear to be characteristic of haemophili. The pathway to succinate in haemophili
appears to be via carboxylation of phosphoenolpyruvate to oxalacetate and thence via malate
and fumarate. The results of tracer studies on a single oral strain of H . parain8uenzae using
various labelled substrates were in keeping with this proposed metabolic pathway.
INTRODUCTION
Haemophili form part of the normal oral flora (Sims, 1970; Kilian, 1976) and are commonly
found in specimens from infections of the teeth and jaws in which they are occasionally the
predominant organisms (Sims, 1974). The haemophili are usually of the V-factor (NAD)requiring species with Haemophilus influenzae being isolated infrequently and capsulated forms
of this species never. The contribution which haemophili make to these oral infections, which
invariably involve a variety of other bacteria, is difficult to determine but we have been
examining the endotoxins, enzymes and various other aspects of these oral haemophili and
comparing them with the established pathogenic capsulated species in the hope of elucidation.
We report here on the acid end-products of metabolism of haemophili and, because the findings
seemed to lack accord with the small amount of published work in this field, some preliminary
studies of enzymes, the incorporation of radioactive substrates into cell constituents and the
conversion of radioactive glucose into acid end-products.
METHODS
Organisms. These were Haemophilus injluenzae, capsulated types a to f, respectively NCTC 8465, 7279, 8469,
8470, 8472 and 8473, H . parainjluenzae NCTC4101 and 10665; and, isolated from various oral sources, H .
injluenzae (non-capsulated) ( 3 strains), H . haemolyticus (3 strains), H . parainfluenzae (4 strains), H .
parahaemolyticus (8 strains), H . aphrophilus (3 strains) and H , paraphrophilus (5 strains).
t Present address: The Dental School, UCL School of Medicine, Mortimer Market, London WClE 6JD, UK.
0022-1287/84/0001-1631$02.00 0 1984 SGM
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1788
I . E . T U Y A U , W . SIMS A N D R . A . D . WILLIAMS
Growth of haemophili.All strains were maintained on heated blood agar incubated at 37 "C in air supplemented
with 5% (v/v) C o t . Haemophili were grown in broth containing per litre: neopeptone (Oxoid), 15 g; yeast extract
(Difco) 5 g; NaC1, 2.5 g; cysteine hydrochloride, 1 g; 5 ml salts solution A (0.5 g each of ZnS0,.4H20,
MnSO, .4H20and FeSO,. 7H20, and 5 mlO.1 M-H2S04per litre); and 0.1 ml salts solution B (1 g MgSO,. 7H20,
1 g (NH4)2S04and 5 g sodium citrate per litre). When required, glucose was added to give a final concentration of
0.5% (w/v). For some experiments, pyruvate, malate, fumarate or 2-oxoglutarate was added to a final concentration of 10 mM and the salts solutions were omitted. Each litre of medium was supplemented with 0.1 ml of a 1%
(w/v) solution of NAD and 0.2 ml of a 1% (w/v) solution of haematin.
Estimation of acid end-products. The general methods, preparation and incorporation of standards were
essentially as described by Holdeman et al. (1977). Broth (5 ml) was inoculated with one drop of an overnight
culture and incubated at 37 "C for 48 h. It was then centrifuged and the supernatant removed. Volatile acids were
extracted by mixing 0.2 ml 50% (v/v) H2S04 with 2.0 ml supernatant, adding 0.4 g NaC1, mixing gently with
1.0 ml diethyl ether, briefly centrifuging to break any emulsion, freezing at - 20 "C and removing the upper ether
layer. The non-volatile acids were extracted by mixing 1a 0 ml supernatant with 0.4 ml50% (v/v) H2S04 and 2.0 ml
methanol and heating the mixture for 30 min at 56 "C. The methylated acids were extracted in 0.5 ml chloroform.
The acids were identified and measured by comparison with similarly processed solutions of standards using
GLC. Uninoculated broths were processed as described and the values obtained were subtracted from the cultures
of haemophili. For estimating succinate production from various substrates, the controls were broths without
substrate but inoculated with haemophili and incubated. Volatile acids were separated on 5 % free fatty acid phase
Chromosorb G (WS DHCS 80-100 mesh) columns and the non-volatile acids on 20% diethylene glycol succinate
on Chromosorb W (HMDS 80-100 mesh) columns at 135 "C. The instrument was a Perkin Elmer F17 using flame
ionization detectors, N2 (30 1 min-l) carrier gas and H2 and air at 16 and 22 lbf in-2 (110.4 and 151.8 kPa),
respectively, for combustion. All of the strains listed were examined on at least three separate occasions - many
other strains of haemophili have been routinely examined once when isolated from oral infections.
Enzyme assays. Haemophili were grown on brain heart infusion heated blood agar for 24 h at 37 "C, harvested
into PBS [Oxoid Dulbecco A pH 7.3 containing (g 1-l): NaCl, 8.0; KC1 0.2; Na2HP04, 1.15 and KH2P04,0.21,
washed and the packed cells resuspended in water to a concentration of about 7% (v/v). The organisms were
disrupted at room temperature by 30 s bursts of sonication, centrifuged at lOOOOg and 4 "C for 30 min and the
supernatant used for the assays. Enzyme activity was determined in 1 cm light path cuvettes containing 3 ml
reaction volumes using a recording spectrophotometer. The methods of Reeves et al. (1971) were used for assaying
citrate synthase (EC 4.1 .3.7), aconitase (EC 4.2.1 .3), isocitrate dehydrogenase (NAD+) (EC 1 . 1. 1.42), 2oxoglutarate dehydrogenase (EC 1 .2.4.2), fumarase (EC 4.2.1 .2), malate dehydrogenase (EC 1 . 1 . 1.37),
isocitrate lyase (EC 4.1 .3,1)and malate synthase (EC 4.1 .3.2). The method of Jurtshuk et al. (1969) was used for
determining succinic dehydrogenase (EC 1.3.99.1) and fumarate reductase (NADH) (EC 1 .3.1.6) was
measured by the method of Kroger et al. (1971). Phosphoenolpyruvate carboxylase (EC 4.1.1.31) and pyruvate
carboxylase (EC 6.4.1.1) were assayed as described by Scrutton (1971). Protein was measured by the Lowry
method using crystalline bovine albumin (Sigma) for the standard.
Incorporation of14Cfrom various substrates. Each substrate [lo pCi (0.37 MBq) U14C] was dissolved to a final
concentration of 2 mM in 150 ml broth which was then sterilized by filtration, inoculated and incubated at 37 "C
for 48 h. After centrifuging at 4 "C and 6000 g for 30 min the cells were washed in distilled water and fractionated
by the method of Roberts et al. (1955). The hydrolysed residue was dried under vacuum over phosphorus pentoxide
and redissolved in 1.0 ml pyridine (0.25 M)/acetate buffer (pH 5.2). Amounts of 25 p1 were separated by
electrophoresis at 700 V and 80 mA for 2 h on Whatman 3MM paper saturated with pyridine/acetate buffer. After
drying, the paper was cut into strips 1.0 cm wide and placed in vials to which 1.0 ml distilled water and 8 ml NE
250 scintillation fluid were added. A Packard Tri-Carb model 3000 liquid scintillation spectrophotometer was
used for counting.
Glucose metabolism. Strain S15 of H. parainjluenzae was grown on brain heart infusion heated blood agar,
harvested into 10 mM-phosphate buffer (pH 7.5) containing 2 mM-ammonium sulphate, washed and resuspended
in the same buffer to an optical density giving about 5 x lo9 organisms per ml. Samples of 1.0 ml were distributed
in vials which were warmed to 37 "C then 0.5 ml 1 .OmM-glucose solution, containing 1 pCi [U-14C]glucose,was
added to each vial and mixed. Vials were kept at 37 "C and removed at 15, 30,45, 60, 75, 105, 120 and 130 min,
acidified with 50 p1 1 M-HCland cooled in melting ice. After centrifuging at lOOOOg for 10 min the Supernatant
was removed and stored at - 20 "C. Organic acids were separated by ascending chromatography on cellulose TLC
(85 : 1 : 14, by vol.) as solvent. This solvent was used because it gave
sheets using butan-1-ol/diethylamine/water
better separation of succinate from acetate and lactate than the other solvents tried [propan-1-ol/ammonia (70 :30,
w/v) and butan-1-ol/acetic acid/water (12 :3 : 5 , by vol.)] although it failed to separate acetate and lactate. The
sheets were dried at 60 "C, cooled, lightly sprayed with 0.02% ninhydrin in acetone warmed to visualize the
ammonium salts of the acids, which were identified by their R, values. Radioactivity was determined in each
sample by cutting the sheets into 1 x 1 cm squares and counting with 5 ml scintillation fluid as above.
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(capsulated)
H . infiuenzae
H . haemolyticus
H . parainfiuenzae
H . parainfuenzae
(NCTC 4101)
H . parahaemolyticus
H. aphrophilus
H . paraphrophilus
H . infuenzae
Species
3
5
0-0.19
04.05
04-23
0.48-2'26
0.99-7.1 3
3-68-5.00
1-02
0-367.32
4.70-7.57
3.58-7-64
0
0-0.07
04-22
1.35
1*60-2-45
6
8
04-05
Succinic
Oxalacetic
04-14
04-59
(rl.52
0
04.11
04-51
0
0
Lactic
Non-volatile acids (mM)
No. of
strains
I
04.30
0
04.29
0
0
04-08
0.74
0
Pyruvic
1
I
04.16
0 4 . 31
04.13
0.48466
0.18-1.77
0-20-2-31
0-95
0.99-2.85
0.22- 1+08
0.18-1-23
04.39
0-37-2.20
0-0.06
0
0
0
Propionic
Acetic
04-06
04-17
0-0-02
0
0
0-0.0 1
04.07
04-08
04.15
04-15
04-05
0
Valeric
0 4 . 01
04.18
04.15
0
iso- or n-
iso- or nButyric
Volatile acids (mM)
Table 1. Acids produced by haemophili when grown in broth containing 0.5% (wIu) glucose for 48 h at 37 "C
\
3
s.
5
%
;s-
s
9
'a
9
3
s
b
I 2
is:
1790
J . E . T U Y A U , W . SIMS A N D R . A . D . WILLIAMS
RESULTS A N D DISCUSSION
The results of a single batch of estimations of the acid end-products of the Haemophilus strains
are shown in Table 1. Succinic acid was consistently the principal acid produced from glucose in
normal broth culture. Acetic acid was also invariably produced but in smaller quantities. Small
amounts of other acids were produced variably from strain to strain. The results obtained
following incubation under strictly anaerobic conditions were essentially the same. Mechanically aerated cultures were considered irrelevant to in vivo conditions and were not studied. The
acids produced by the individual species were not sufficiently dissimilar to be of value in
classification but the general production of succinate and acetate does seem to characterize the
genus. In this respect haemophili resemble Bacteroides, a genus which, interestingly, has DNA
G + C compositions extending over a similar range (Kilian, 1976; Holdeman et al., 1977),
requires haemin for growth and has endotoxins which do not contain 3-keto-deoxyoctonic acid
(Hofstad, 1974; Tuyau & Sims, 1983). Table 1 shows that H.parainfluenza NCTC 4101 produced
much less succinate than the other strains of H . parainfluenzae and was the only strain of
Haemophilus to form significant amounts of oxalacetate and pyruvate, the former being its major
product. Hollander (1976) found all the enzymes of the tricarboxylic acid cycle present in this
strain and thought the cycle to be functional in strain Bossy No. 7 Leidy of H . parainfluenzae
which was studied by White (1966). This strain could not be obtained for study but Hollander
(1976) thought it atypical because of its rapid utilization of glucose with consequent high growth
yield and its feeble malate dehydrogenase activity. It seemed likely that the differences between
Table 2. Enzyme activities of extracts of various haemophili
Enzyme activity [nmol substrate used (mg protein)-' min-l at 25 "C]
I
Strain
1
Succinic
dehydrogenase
Fumarase
Malate
dehydrogenase
Phosphoenol
pyruvate
carboxylase
6.6
8.8
543
62.9
19.9
0.44
28 1
48.2
617
22.4
2040
18.5
H . influenzae
(capsulated NCTC 7279)
H. influeltzae
(oral strain S9)
H. parahaemolyticus
(oral strain S43)
H. parainfluenzae
(NCTC 10665)
H . parainfluenzae
(oral strain S15)
H. parainfluenzae
(oral strain S24)
H. parainfluenzae
(NCTC 4101)*
38.7
6.3
33.0
0.43
34.8
30.4
3128
20.2
10.5
22.4
2095
16.4
27.2
26-8
251 1
18.9
* This strain only had the following enzyme activities also (see text): citrate synthase, 3.18; aconitase, 17.4;
isocitrate dehydrogenase (NAD+), 12.0; 2-oxoglutarate dehydrogenase, 22.3 ; fumarate reductase, 38.9.
Table 3. Succinate produced from various substrates by strains of haemophili
Succinate (mM) produced in 48 h from 10 mM substrate
Strain
Substrate
H . influenzae
(capsulated NCTC 7279)
H. parahaemolyticus
(oral strain S43)
H . parainfluenzae
(oral strain S15)
H . parainfluenzae
(NCTC 4101)
. ..
I
1
Pyruvate
Malate
Fumarate
1*06
0.89
1*40
0
0.69
1.14
0.82
0
0.53
1.32
1.01
0
0.60
0.60
3.50
2-0
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2-Oxoglutarate
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x Total c.p.m. incorporated into cells
Percentage of total c.p.m. incorporated into:
cold TCA-soluble
ethanol-soluble
ether/ethanol-soluble
hot TCA-soluble
hydrolysed residue
x Total c.p.m. in hydrolysate residue
Percentage of c.p.m. of hydrolysate incorporated into:
lysine
glucosamine
neutral compounds
glutamate
aspartate
46-7
4.7
18-8
6.9
75-0
5.5
40.0
1-8
2-7
26.0
3.4
-
0-5
3.8
-
3-8
1-6
5-0
14.5
75.1
114
7-4
9.9
1-8
8-2
72.8
28-3
11.1
30.9
2-9
31.7
23-4
65-8
152
Aspartate
38-9
Glutamate
28 1
Glucose
0-5
4.4
85.4
0.3
0.1
0-9
7-3
69-5
84-5
m.5
1-7
121
Alanine
A
Substrate (2 m ~ )
-, No counts higher than background detected.
f
55-9
1-4
7.3
-
1.9
8-1
21-5
2-4
23-2
44-6
13-3
29-8
Lactate
46-9
2-1
13-2
-
1.3
7.2
56-5
2-4
13-2
20.7
17.0
32-3
Malate
Table 4. Fractionation of H . paramjluenzae S15 after growth in broth containing I4C-labelled substrates
0-5
90.7
0-2
0.7
-
3.1
88.7
0-3
2.3
5.5
12-7
229
\
Acetate
%s
4
3-
b
5
B
kY
9
3
b
5
1792
J . E . T U Y A U , W . SIMS A N D R . A . D . WILLIAMS
strain NCTC 4101 and the other haemophili studied would involve enzymes of the tricarboxylic
acid cycle.
The enzyme activities of some strains of haemophili are shown in Table 2. Although we were
able to confirm the finding of Hollander (1976) that strain NCTC 4101 has all the enzymes of the
tricarboxylic acid cycle, the strains examined by us had only four enzymes of the central
metabolic pathways related to the cycle, namely, succinic dehydrogenase, fumarase, malate
dehydrogenase and phosphoenolpyruvate-carboxylase. A partial tricarboxylic acid cycle and
high malate dehydrogenase activity seem to be characteristic attributes of most haemophili. All
of the haemophili tested lacked the enzymes isocitrate lyase, malate synthase and pyruvate
carboxylase, which excludes the glyoxalate bypass or direct conversion of pyruvate to
oxalacetate in these organisms. Since fumarate reductase was not detected, fumarate is
presumably reduced by succinic dehydrogenase. Warringa et al. (1958) demonstrated the
reversibility of succinic dehydrogenase and its physiological importance for an oral anaerobic
coccus.
All the strains of haemophili tested produced succinate in broth containing pyruvate, malate
or fumarate instead of glucose (Table 3). In contrast, only strain NCTC 4101 formed succinate
in the presence of 2-oxoglutarate. It could be that the other strains were impermeable to 2oxoglutarate but the enzyme assays and the results of the experiment with radioactively labelled
substrates suggest this is due to an incomplete tricarboxylic acid cycle.
The probable pathway in haemophili is carboxylation of phosphoenolpyruvate to oxalacetate
by phosphoenolpyruvate-carboxylase,then via malate and fumarate to succinate catalysed by
malate dehydrogenase, fumarase and succinic dehydrogenase, respectively. Acetate would
presumably be formed from pyruvate via acetyl-CoA. The pattern of incorporation into cell
constituents of [14C]glucose, amino acids and carboxylic acids by growing cells of H .
parainflzienzae S15 (Table 4) is revealing, particularly with respect to the relative labelling of
aspartate and glutamate. Glucose carbon was widely distributed, about 34% entering the lipid
fraction via acetate and fatty acid synthesis. [ 14C]Acetate itself was 89% incorporated into
lipids, with negligible labelling of aspartate and glutamate. The 3C substrates alanine and
lactate, each convertible to acetate via pyruvate, gave similar fractions of their incorporation to
the lipids although the extent of incorporation was quite different. With alanine much of the
incorporation was into proteins, like the other amino acids aspartate and glutamate. Negligible
label from alanine flowed into aspartate or glutamate and the largest fraction of radioactivity
from either aspartate or glutamate was directly incorporated into protein. Those substrates
which labelled the dicarboxylic amino acids significantly all formed aspartate more than
glutamate in the ratios 4.5 : 1 for glucose, 5 : 1 for lactate and 7 : 1 for malate. It is clear that
glutamate can be metabolized by haemophili but these results support the enzymological
findings that such metabolism is not via the tricarboxylic acid cycle.
t
20
40
60
80
100
120
140
Time (min)
Fig. 1. Time-course for the conversion of radioactively labelled [U-14C]glucose into succinate and
acetate/lactate by an oral strain (S 15) of Haemophilus paruinfluenzue. A,Succinate formed ; 0 ,acetate
or lactate formed; I,
glucose used.
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Acid end-products of haemophili
1793
Figure 1 summarizes the results of a time-course conversion of 0.33 mM-glucose into
succinate, acetate and lactate by a washed cell suspension of H . parainjluenzae S15. Acetate and
lactate could not be separated by the TLC system used which was chosen for its clear separation
of succinate from other metabolites. The metabolism over 2 h led to 75% of glucose being
converted to these three acids with 25 % incorporation. This shows that these metabolites, which
were measured in spent broth, can be formed from glucose in large quantities and are unlikely to
have been produced from other media constituents.
Although these investigations of Haemophilus metabolism are only preliminary and
exploratory, it seems reasonable to suggest that haemophili produce succinic acid as a major
end-product of metabolism because many strains lack some of the enzymes of the tricarboxylic
acid cycle.
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R. (1976). Energy metabolism of some
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