Download Effects of oxygen on the growth and metabolism of Actinomyces

FEMS Microbiology Ecology 53 (1988) 45-52
45
Published by Elsevier
FEC 00147
Effects of oxygen on the growth and metabolism
of A ctinomyces viscosus
M.H. De Jong, J.S. Van der Hoeven, C.W.A. Van den K i e b o o m and P.J.M. Camp
Department of Preventive and Community Dentistry, University of Nijmegen, Nijmegen, The Netherlands
Received 26 June 1987
Revision received 17 August 1987
Accepted 19 August 1987
Key words: Actinomyces; Oxygen; Cytochrome; Dental plaque
1. SUMMARY
Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral
Streptococcus spp., a saccharolytic and aerotolerant organism. We have investigated the effects of oxygen on the growth and metabolism of
A. viscosus. To this end A. viscosus Ut 2 was
grown in a glucose limited chemostat culture on a
chemically defined medium (D = 0.2 h -1) with
exposure to variable amounts of oxygen. The
Y~u¢o~ increased from 62.5 g.mo1-1 under
anaerobic conditions to 149 g.mo1-1 under
aerobic conditions, while, concomitantly, the
carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of
[14C]glucose to the chemostat showed that the
glucose, which was not converted to acidic fermentation products, was instead converted to
carbon dioxide or used for the production of
biomass. Under aerobic and anaerobic conditions
identical cytochrome spectra, containing only two
cytochrome b-type absorbtion bands, were found.
Correspondence to: J.S. van der Hoeven, Department of Preventive and CommunityDentistry, University of Nijmegen,
P.O. Box 9101, 6500 HB Nijmegen,The Netherlands.
It was concluded that electron transport phosphorylation probably occurs both under aerobic
and anaerobic conditions. Anaerobically, fumarate
served as the electron acceptor, while the high
growth yields observed under aerobic conditions
are likely to be explained by citric acid cycle
activity coupled to electron transport phosphorylation.
2. INTRODUCTION
Actinomyces viscosus, Actinomyces naeslundii,
Streptococcus mitior and Streptococcus sanguis are
pioneers in starting dental plaque formation and
remain the predominant microorganisms in
supragingival plaque at healthy sites [1,2]. These
species are carbohydrate fermenters tolerant to
oxygen. Recently we have shown that S. mutans
and S. sanguis [3] consume considerable amounts
of oxygen by the partial oxidation of sugars to
acetic acid, carbon dioxide and water. Oxygen
consumption proceeds only at low dissolved
oxygen concentrations and is apparently not coupled to electron transport phosphorylation, since
the growth yield of the streptococci was only
slightly affected [3]. Similarly, Lactobacillus
0168-6496/88/$03.50 © 1988 Federationof EuropeanMicrobiologicalSocieties
46
plantarum can grow at the expense of lactate
under aerobic conditions, but its metabolism remains coupled to substrate level phosphorylation
[4].
Anaerobically A. viscosus and A. naeslundii
ferment glucose according to Schemes 1 and 2
[5,6]. Under aerobic conditions, in batch culture,
glucose is partially oxidized according to Scheme
3 [5].
(1) glucose ~ 2 lactic acid
(2) glucose + carbon dioxide -~ formic acid +
acetic acid + succinic acid
(3) glucose + 2 oxygen ~ 2 acetic acid + 2
carbon dioxide + 2 water
The high growth yields [6] reported for succinate-producing cells (Scheme 2; Ygl.... e = 65 g.
mo1-1) strongly suggest that in A. viscosus the
production of succinate [7] is coupled to electron
transport phosphorylation [6]. The high growth
yield (Yglu¢o~= 87 g. mol-1) reported for aerobic
batch cultures [5] suggests that electron transport
phosphorylation might also occur under aerobic
conditions. In Propionibacterium spp. another
group of anaerobes tolerant of oxygen, cytochromes are present and electron transport phosphorylation has been shown to occur under aerobic and
anaerobic conditions [8,9]. However, the occurrence of cytochromes or oxidative phosphorylation in A. viscosus has never been investigated.
We have therefore studied the effects of oxygen
on the growth and glucose metabolism of A.
viscosus and have studied the presence of cytochromes in aerobically and anaerobically grown
cells.
3. MATERIALS AND METHODS
3.1. Growth conditions
A. viscosus Ut 2, originally isolated from human dental plaque [10], was grown on a filtersterilized chemically defined medium [11], which
contained amino acids, nucleotides, vitamins, inorganic salts, trace elements, a bicarbonate buffer
and 10 mM glucose to give carbohydrate limitation. The bacteria were grown at a dilution rate of
D = 0.2 h-1 at 37°C in a chemostat (500 ml) as
described before [3]. The pH was kept at 7.0 by
automatic titration with 2 N KOH. The appropriate gas phase was obtained by mixing N 2
(95%), CO 2 (5%), with N 2 (75%), CO2 (5%) and 02
(20%). The filter-sterilized gas was sparged into
the medium (5 1. h -1) below the stirrer (100 rev.
rain-l). The purity of the culture was routinely
checked on aerobically and anaerobically incubated blood agar plates. The identity of representative isolates was routinely checked with the
API 20A system.
3.2. Analytical procedures
Dry weights were determined in 40-ml samples
taken directly from the culture by syringe. The
samples were centrifuged and washed three times
in demineralized water. The cells were dried in
crucibles at 105°C until they reached constant
weight. Dry weight determinations were done at
least in duplicate in separate cultures. Formic,
acetic, succinic, lactic, citric and glutamic acids
were determined with isotachophoresis [12]. Spectra of dithionite-reduced cytochromes in whole
bacterial cells were obtained with an Aminco
Chance spectrophotometer (American Instruments
Co., Washington, U.S.A.) [13]. Aerobically grown
cells (1 mg. m1-1 dry weight) were centrifuged,
resuspended in chemically defined medium
without glucose and incubated with glucose (2
mM), acetate (10 raM), formate (10 raM) and
succinate (10 mM). The rates of oxygen consumption and the K m value for oxygen (Gottschal, J.C.,
personal communication) were determined in these
cell suspensions using a polarographic oxygen
electrode.
3.3. Metabolism of [ ] 4C]ghlcose
170/~Ci of uniformly labelled [14C]glucose (270
mCi- mmol-1) was added to a chemostat (500 ml)
with a culture of A. viscosus in steady state (D =
0.19 h - l ; pH 7.0). The culture was gassed with a
mixture of N 2 (80.4%), CO 2 (5%) and 0 2 (14.6%);
the dry weight was 1.30 mg- ml-1. At regular time
intervals after the addition of [14C]glucose 1.0-rftl
samples were taken directly from the culture by
syringe and added to 0.1 ml KOH. These samples
were centrifuged immediately at 20 °C for 4 min
at 18 400 × g. The pellet fraction was washed once
and subsequently resuspended in 1.0 ml water.
47
Two 0.1 ml portions were taken from the supernatant fraction. The radioactivity in one portion
was counted directly, while 0.1 ml 1.0 M N a H C O 3
and subsequently 0.1 ml 2.0 N HC1 were added to
the other portion before counting radioactivity.
The latter procedure decreased the p H below 1.0
and removed all 14C-labeled carbon dioxide produced by the bacteria. The radioactivity in 0.1 ml
portions of the pellet and the two supernatant
fractions was counted in 10 ml Aqualuma plus
(Baker). The amount of 14C-labeled carbon dioxide in the culture fluid was calculated by substraction of the amounts of radioactivity in the
two supernatant fractions after correction for the
dilution. In addition the 14C-labeled carbon dioxide evolving from the culture fluid into the gas
phase was captured in three successive washbotties containing 1 N K O H and were counted separately.
only parts of RNA and D N A synthesized from
glucose were ribose and deoxyribose, and that all
amino acids required for protein synthesis were
derived from the medium and not synthesized
from glucose. The main phospholipid in the cells
was assumed to be phosphatidylethanolamine with
two C16 fatty acids. Glycerolphosphate and the
two C16 fatty acids were assumed to be synthesized from glucose, the latter two via acetyl-CoA
[14]. The fraction of the molecular weight of the
monomers derived from glucose was calculated as
follows: number of C-atoms derived from glucose
per monomer ×12, divided by the molecular
weight of the monomer. To obtain the amount of
glucose-carbon incorporated into 100 g of biomass
(dry weight), these fractions were multiplied by
the amount of the respective cellular constituents
per 100 g of biomass (see Table 1).
3.4. Calculations
4. RESULTS
The amount of glucose-carbon incorporated in
the biomass was calculated on the basis of data
given by Stouthamer [14] for the composition of
microbial cells (see Table 1). It was assumed that
all polysaccharides (assumed to be polyglucose)
were synthesized directly from glucose, that the
A . viscosus was grown in a glucose-limited
chemostat on a chemically defined medium (D =
0.2 h -1) under exposure to variable amounts of
oxygen. The effects of increasing concentrations
of oxygen in the gas phase on the growth yield,
Fermentation products
1 5- (mot.mo1-1 glucose)
10"
/ / /-1
x~---~. T
[
Q5.
o
t'o
%0 2
Fig. 1. The effect of increasing concentrationsof oxygenin the gas phase on the fermentation products of A. viscosus growing under
glucose limitation (D = 0.2 h-l). C), succinic acid; I, acetic acid; zx, formic acid.
48
_ _
% Glucose - C incorporated
100
'°
......
Dry weight (g cells mo1-1 glucose)
~
150
*
.
100
6O
o
50
i
~
0
i
I
I
10
15
20
%o 2
Fig. 2. Effect of increasing concentrations of oxygen in the gas-phase on the dry weight (g cells m o l - l glucose) and on the partition
of glucose carbon (% glucose-C incorporated) over the biomass (calculated from the dry weight with the aid of Table 1), carbon
dioxide (calculated) and acidic fermentation products (measured with isotachophoresis) of A. viscosus growing glucose limited at
D = 0.2 h -1. * , dry weight; e, % glucose-carbon in the biomass; I , % glucose-carbon in the acidic fermentation products; O, %
glucose-carbon in carbon dioxide.
fermentation products and carbon recovery are
shown in Figs. 1 and 2. Under anaerobic conditions formic, acetic and succinic acid were produced in almost equimolar amounts according to
Table 1
The amount of glucose carbon incorporated in microbial cells,
growing under glucose limitation on a chemically defined
medium containing all amino acids, nucleotides, vitamins, salts
and trace elements
Macromolecular
substance
Amount
(g/per
100 g
cells)
Mean
molecular
weight
monomer
Number of
glucose
carbons
incorporated
Polysaccharide
Protein
Lipid
RNA
DNA
16.6 a
52.4
9.4
15.7
3.2
162
690
300
280
6 (0.44) b
0 (0.00)
37 (0.64)
5 (0.20)
5 (0.21)
Total
97.3
Amount of
glucose
carbon
(g carbon
100 g cells)
7.38
0.0
6.05
3.14
0.69
17.26
a Data from Stouthamer [14].
b In parenthesis: fraction of the molecular weight of monomer
derived from glucose carbon.
Scheme 2. At increasing oxygen concentrations
first succinic acid disappeared with a concomitant
increase in the amounts of formic and acetic acid
produced (Fig. 1). Beyond 5% oxygen the amounts
of formic and acetic acid also decreased, until at
20% oxygen in the gas phase these acids were no
longer produced. No other fermentation products
such as ethanol and pyruvic, lactic, citric or
glutamic acids were found (results not shown). In
washed cell suspensions of aerobically grown cells
oxygen was consumed at a considerable rate (101
nmol O 2- mg -1 dry weight, min -1) and with a
high affinity ( K m = 1 # m o l . l - 1 ) , when glucose
(2.0 m m o l . 1 - 1 ) was added as an external substrate. Addition of acetic, formic and succinic
acids to these cell suspensions did not result in
oxygen consumption.
The Yglucose first increased rapidly from 62.5
g - t o o l -] under anaerobic conditions, to 122.5
g. mo1-1 at 8% oxygen and subsequently more
slowly to 149 g. mol-1 at 20% oxygen. The calculations in Table 1 show that a fixed percentage of
the biomass was derived from glucose carbon,
consequently the amount of glucose carbon incor-
49
Table 2
Log
6
com ..
Partition of glucose carbon
Incorporated in
Biomass
Fermentation products
Carbon dioxide
Percentage glucose carbon
[14C]Glucose
Calculated a
35.0
32.7
32.3
31.2
32.8
36.0
a Dry weight used for the calculation of the amount of glucose
carbon incorporated into the biomass (see Table 1 and Fig.
2) was from a sample taken 1 day before the addition of
[14C]glucose; fermentation products were determined in the
same sample using isotachophoresis. The remaining glucose
carbon, which was not incorporated into the biomass nor
into the acidic fermentation products, was assumed to have
been converted to carbon dioxide.
porated into the biomass increased proportionally
with the yield (Fig. 2). The a m o u n t of glucose
c a r b o n recovered from the fermentation products
decreased, however, f r o m 75% under anaerobic
conditions, to only 7% at 20% oxygen. The remaining glucose carbon, neither incorporated into
biomass nor into acidic fermentation products,
was supposed to be converted to c a r b o n dioxide
(Fig. 2).
I n order to verify whether the partition of
glucose c a r b o n over biomass, acidic fermentation
p r o d u c t s and c a r b o n dioxide, as depicted in Fig. 2
was correct, 170 # C i of uniformly labelled
[14C]glucose were added to a chemostat culture.
D u r i n g the experiment the oxygen level in the gas
phase was 14.6% and the dry weight was 130
r a g . ml-1. The results are shown in Fig. 3. Linear
regression of the log 14C counts towards t = 0
gave the a m o u n t s of glucose c a r b o n incorporated
into the biomass, acidic fermentation products
a n d c a r b o n dioxide. The w a s h o u t rates of
[14C]carbon in the biomass and acidic fermentation p r o d u c t s were 0.18 h - t and 0.19 h -1, respectively, and in g o o d agreement with the imposed
dilution rate of the culture (0.19 h - l ) . The
[14C]carbon dioxide dissolved in the culture fluid
h a d a m u c h higher washout rate (1.4 h - l ) . This
was p r o b a b l y due to exchange of dissolved c a r b o n
dioxide with c a r b o n dioxide f r o m the gas sparged
t h r o u g h the culture fluid. Accordingly the a m o u n t
o f 1 4 C 0 2 captured in the washbottle with K O H
~o
6
-c 0 2
i
6
,0
2'0
- - -
i
~0
hours
Fig. 3. Metabolism of [14C]glucose. Log cpm in the biomass
(v P), in the acidic fermentation products (e, S) and in carbon
dioxide (O, CO2) at different time intervals after the addition
of [lac]glucose to a continuous culture of A. viscosus growing
glucose limited under 14.6% oxygen (D = 0.19 h-l).
was identical to the a m o u n t lost from the culture
fluid (results not shown). In Table 2 the data for
the partition of 14C over the various fractions are
c o m p a r e d with the data derived from chemical
analyses of the fermentation products and the
calculated a m o u n t s of glucose-carbon in the bioTable 3
b-type cytochromes in aerobically and anaerobically grown
cells of A. viscosus
Computer analysis of the observed absorbtion spectrum showed
that the spectrum consisted of two absorbtion bands at two
different wavelengths (I and II) [13]. The two absorbtion bands
had different intensities, which were expressed as the % area of
the observed absorbtion spectrum.
anaerobe
Wavelength
% Area
aerobe
I
II
I
II
558.6
72.5
564.3
27.6
558.0
73.7
563.5
26.4
50
mass and carbon dioxide. The data are in good
agreement, with the 14C method giving a slightly
higher value for the biomass and a somewhat
lower one for carbon dioxide.
At high percentages of oxygen in the gas phase,
a high Ygl..... (149 g. mol-1), combined with a
low carbon recovery from fermentation products
was observed (Fig. 2). This strongly suggested an
oxidative metabolism coupled to electron transport phosphorylation. We have therefore investigated the presence of cytochromes in cells grown
under aerobic and anaerobic conditions. In the
dithionite-reduced cytochrome spectra only two
absorbtion bands of b-type cytochromes could be
detected by computer analysis [13]. Their ratios
remained the same under aerobic and anaerobic
conditions (Table 3).
5. DISCUSSION
A. viscosus, an organism generally considered
as an anaerobe, grew with a 2.4-times higher yield
in aerated cultures than under anaerobic conditions (Fig. 2). The growth yield in aerated cultures
(Yglucose= 149 g-mo1-1) cannot be accounted for
by a fermentative metabolism and strongly suggested citric acid cycle activity coupled to electron
transport phosphorylation, as was observed before
in other oxygen consuming anaerobes, such as
Propionibacterium pentosaceum [15]. This suggestion was confirmed by the observation that with
increasing oxygen concentrations carbon dioxide
became almost the sole metabolic product, while
acetate, formate and succinate disappeared concomitantly (Figs. 1 and 2, Table 2). Moreover, in
the closely related species A. naeslundii, the citric
acid cycle enzymes have been demonstrated [5],
yet the hnk to oxidative catabolism in Actinomyces
species has not been made. Taken together the
above strongly suggests that A. viscosus adjusts its
metabolism to the amount of oxygen available in
the environment. It can grow either completely
fermentatively (Schemes 1 and 2), or via partial
(Scheme 3) or complete oxidation of carbohydrates
(Scheme 4).
(4) glucose + 6 oxygen ~ 6 carbon dioxide + 6
water
The cytochrome spectra of A. viscosus cells
grown under aerobic and anaerobic conditions
were identical: only two cytochrome b-type
absorbtion bands were present (Table 3). Under
anaerobic conditions these cytochromes are presumably part of an electron transport chain
terminating with fumarate reductase [16], which
enables the production of 1 mol of ATP per mol
succinate formed [17]. Fermentation of 1 mol glucose would then yield 4 mol ATP. Since, with
glucose as an electron donor oxygen was consumed with a high rate and affinity, it seems
reasonable to assume that under aerobic conditions a very similar electron transport chain is
active, now with oxygen as the final electron
acceptor. If again, as under anaerobic conditions,
1 ATP would be produced per 2 e- passing the
electron transport chain, complete oxidation of
glucose would yield 14 mol of ATP. Taking into
account the fraction of glucose converted into
biomass and the partial oxidation of glucose, an
YATP value of 18.4-20.8 g. mo1-1 under anaerobic
conditions and an YATP of 18.0--21.8 g-mo1-1
under aerobic conditions can be calculated. These
values are in good agreement with each other, but
somewhat higher than the YATP values observed
for propionic acid bacteria (11.5-16.7 g-mo1-1)
growing anaerobically with glucose or fructose [9].
The calculations in Table 1 show that, for a
growth medium with a specific composition, a
fixed percentage of the dry weight of the cells is
derived from glucose. As a consequence, with increasing growth yields, an increasing part of the
glucose would be used for the production of biomass. This was verified in the experiment in which
[14C]glucose was added to the chemostat. The
observed incorporation of [14C]glucose into the
biomass was in good agreement with the calculated amount (Table 2). In addition the isotope
experiment showed that the remaining glucose
carbon, which was not incorporated into the biomass or acidic fermentation products, was indeed
converted to carbon dioxide.
In conclusion the present experiments show
that A. viscosus grows as well in aerated cultures
as under anaerobic conditions. Oxidation of glucose was presumably coupled to electron transport
phosphorylation, which enabled A. viscosus to
51
i n c r e a s e its g r o w t h y i e l d u n d e r a e r o b i c c o n d i t i o n s
significantly. Competing microorganisms from the
s a m e e c o s y s t e m , s u c h as S. sanguis, s h o w s i m i l a r
r a t e s of o x y g e n u p t a k e [18], b u t t h e y c a n n o t significantly increase their growth yield under aerobic
c o n d i t i o n s [3]. T h i s m i g h t b e a c o m p e t i t i v e adv a n t a g e f o r A . viscosus in d e n t a l p l a q u e , w h e r e
o n l y l i m i t i n g a m o u n t s o f c a r b o h y d r a t e s [19] a n d
o x y g e n [20] a r e a v a i l a b l e .
ACKNOWLEDGEMENT
W e w i s h to t h a n k L . F . O l t m a n a n d W . d e V r i e s
f o r t h e i r p r e p a r a t i o n o f the c y t o c h r o m e s p e c t r a ,
and stimulating discussions.
[10]
[11]
[12]
[13]
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