FEMS Microbiology Letters 16 (1983) 69-74
Published by Elsevier Biomedical Press
69
The relation between growth rate and electrochemical proton
gradient of Streptococcus cremoris
Roel Otto, Bart ten Brink, H. V e l d k a m p and Wil N. K o n i n g s
*
Department of Microbiology, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
Received7 July 1982
Accepted 8 July 1982
1. I N T R O D U C T I O N
It is now generally accepted that the electrochemical proton gradient (Ak~H+) or proton-motive force (pmf) plays an important role in the
metabolism of microorganisms. It is the main driving force for energy-requiring processes, such as
ATP synthesis and secondary solute transport
across the cytoplasmic membrane [1,2]. Recently,
it was shown that the A/~H+ is also involved in
other metabolic processes, such as nitrogen fixation [3], ppGpp breakdown [4] and DNA-uptake
by genetic transformation [5]. Although there exists a considerable knowledge about the role of the
A/~H~ in several specific metabolic events, surprisingly little is known about the A/~H+ in relation to
the overall process of microbial growth. Besides
ATP, which acts as a molecular currency of metabolic energy the A~ H÷ is in many cases an obligate
intermediate between energy-consuming and energy-producing processes. A complete description
of the energetics of microbial growth will only be
possible if during growth the A~H+ of microorganisms can be recorded.
In this report we present the results of a study
on the magnitude and composition of the Ak~H~ of
Streptococcus cremoris growing in continuous cultures at different rates and different pH values.
* To whom correspondenceshould be addressed.
The strictly fermentative lactic acid bacterium S.
cremoris was chosen because of its very simple
metabolism; ATP is produced only by substrate
level phosphorylation (exclusively from carbohydrates) and a A/2H+ is generated either by H ÷
extrusion via the membrane-bound CaZ+/Mg 2+stimulated ATPase or by export of lactate [6].
Estimations of the A/~H+ in another lactic streptococcus species, S. lactis, have been reported, both
in resting cells [7] and recently in cells growing in
batch culture [8]. The results of the latter study
prompted the author to suggest that the magnitude
of the A/iH~ of S. lactis was independent of growth
rate. This conclusion was mainly based on two
observations (i) the growth rate of S. lactis on
glucose was independent of the chemical composition of the growth medium although this resulted
in drastic differences in the A/~H+, and (ii) the
A/2H+ was not affected by varying the growth rate
by adding different fermentable carbohydrates to
the medium.
However, it must be emphasized that in batch
cultures the chemical composition of the growth
medium changes continuously as growth proceeds,
and these changes will affect the physiology of the
organism. It is therefore more convenient to study
the relation between the pmf and growth rate in
chemostat cultures. This type of culture offers the
advantage, that microorganisms can be cultivated
at different growth rates in media of constant
chemical composition [9]. Our results show that
0378-1097/83/0000-0000/$03.00 © 1983 Federation of European MicrobiologicalSocieties
7o
the AtiH+ of S. cremoris varies inversely with
growth rate.
2. MATERIALS A N D M E T H O D S
Culture conditions. S. cremoris Wg2 was obtained from the Dutch Institute of Dairy Research
(Nederlands Instituut voor Zuivelonderzoek
(NIZO), Ede, The Netherlands). The organism was
routinely maintained in 10% (w/v) sterile skimmed
milk, and stored at - 2 0 ° C until use. From the
milk cultures S. cremoris was transferred to a
complex MRS medium [10] and subsequently to a
chemically defined medium which contained per
litre distilled water: 2.5 g lactose, 2.5 g K 2 H P O 4,
3.0g KH2PO4, 0.6g (NH4)3-citrate, 1.0g Naacetate, 0.25 g cysteine-HC1, 5.0g salt-free, vitamin-free casein hydrolysate (ICN Pharmaceuticals
Inc., Cleveland, OH), 10 ml vitamin solution, 10
ml metal solution, 10 ml nucleic acid bases solution. The vitamin solution contained per litre distilled water: pyridoxine-HC1, 200 mg; nicotinic
acid, 100 mg; thiamin-HC1, 100 mg; riboflavin,
100 mg; Ca-(D+)pantothenate, 100 mg; Nap-aminobenzoate, 1 g; D-biotin, 1 g; folic acid, 100
mg; vitamin B~2, 100 mg; orotic acid, 500 mg;
2-deoxythymidine, 500 mg; inosine, 500 mg; DL6,8-thioctic acid, 250 mg; pyridoxamine-HC1, 500
mg. The pH of this solution was 7.0. The metal
solution contained per litre distilled water: MgC12
-6H20, 20g; C a C l z . 2 H 2 0 , 5g; F e C I 2 . 4 H 2 0 ,
0.5 g; ZnSO4.7H20, 0.5 g; CoC12 • 6H20, 0.25 g.
The nucleic acid bases solution contained per 10
ml 0.1 N NaOH: adenine, 10 mg; uracil, 10 mg;
xanthine, 10 mg; guanine, 10 mg. The pH was
adjusted to pH 6.6 with 1 N NaOH. The medium
was sterilized by passage through a cellulose nitrate
membrane filter (0.15 /~m, Sartorius, Gi3ttingen,
F.R.G.). Chemostat cultures were grown anaerobically under N 2 atmosphere in glass fermentors
with a working volume of 170 ml at 30°C and
controlled pH of 5.7; 6.4; 7.0 or 7.6 as described
previously [6].
2.1. Measurement of p H gradient (ApH) and membrane potential (A~p)
The Aq~ and ApH values were calculated from
the accumulation of [3 H]-tetraphenylphosphonium
ion (Ph4 P+) and [14C]benzoic acid, respectively.
The accumulations of Ph4 P÷ and benzoate were
determined by adding 6 nM [3H]PhaP+ and 0.13
/~M [14C]benzoate (final concentrations) to an actively growing culture of S. cremoris (approx. 300
mg cell dry weight/litre). After a 5-min period of
incubation, 10-ml samples from the culture were
collected in calibrated test tubes and quickly
filtered over cellulose acetate filters (0.45 /~m;
Schleicher and Schtill, Dassel, F.R.G.). Filters were
transferred to counting vials and the radioactivity
was measured with a liquid scintillation counter.
The binding of Ph4 P÷ to cell constituents was
estimated after de-energizing the cells by treatment with 1% toluene or 10% butanol for 1 h at
room temperature. Both procedures gave essentially the same results. Binding of Ph4 P÷ and
benzoate to cellulose acetate filters was insignificant.
2.2. Intracellular volume
The internal volume was determined from the
distribution of 3H-labelled water and [14C]taurine
by the procedure described by Bakker et al. [11].
For chemostat-grown cells the intracellular volume
was 3.8 /~l/mg of cell protein. This volume was
approximately constant (-+0.4 ~ l / m g protein) at
all growth rates tested.
2.3. Analysis of metabolic end products
Lactate was determined in the cell-free culture
fluid as described previously [6].
2.4. Protein
Protein was determined by the method of Herbert et al. [12].
2.5. Materials
[t4C]Taurine (56 Ci/mol) and [14C]benzoic acid
(56 Ci/mol) were obtained from the Radiochemical Centre (Amersham, U.K.). [3H]Ph4 P+ (2500
Ci/mol) was obtained from the Nuclear Research
Centre Negev, Beer Sheva, Israel. All other chemicals were of the highest purity available and
purchased from commerical sources.
71
A
i
B
100
E
100
120
E
9<3
E
J
.<
8O
3-
lOC
Ill
I-0
D-
E
2t (rain)~
100
LU
Z
t (min)
W
(.D
n,0
LI_
rr
m
Ill
o
•
o
0
50
5C
10
20
30
z.0
50
60
t (mini
10
20
30
Z,0
50
60
t (rain)
Fig. 1. (A) and (B) The membrane potential and ,fipH in S. cremoris during lactose starvation. S. cremoris was grown in a lactose
limited chemostat at pH 5.7 and pH 7.0 at a dilution rate of 0.15/h. The A+ (~-and zipH (0 were determined by adding a small
amount of [3H]Ph4P+ and [t4C]benzoic acid to the culture. After 5 min incubation in situ the culture was harvested and at regular
time intervals samples of 5 ml were taken and quickly filtered. Zero time was the moment of harvesting, filtration of the first sample
took 10-15 s (first point in the graph). The experiments were performed at two different pH values. (A) pH 5.7; (B) pH 7.6. The inset
shows the time course of the decay of the membrane potential at an extended time scale.
3. R E S U L T S
The theory of c o n t i n u o u s culture predicts that
the c o n c e n t r a t i o n of the growth-limiting n u t r i e n t
i n a chemostat becomes very low u n d e r steady-state
c o n d i t i o n s [9]. I n our c o n t i n u o u s culture experim e n t s lactose was the growth-limiting n u t r i e n t and
this could not be detected in the culture fluid
u n d e r steady-state conditions. W h e n samples are
w i t h d r a w n from the culture the last traces of lactose
in the growth m e d i u m will be rapidly consumed.
Lactic streptococci convert lactose for more than
95% to lactic acid a n d this fraction represents the
energy source. The r e m a i n i n g 5% is incorporated
into structural cell material. Lactose starvation will
therefore rapidly lead to energy starvation a n d
c o n s e q u e n t l y rapid changes of the c o m p o n e n t s of
the p r o t o n - m o t i v e force m a y occur. Since our goal
was to measure the c o m p o n e n t s of the p m f in
"growing" S. c r e m o r i s a n d the i n t r o d u c t i o n of a
short starvation period d u r i n g the course of sampling a n d filtration was u n a v o i d a b l e we investigated whether the m a g n i t u d e and the compositions of the p m f did not change d u r i n g the sam-
72
pling period. For that purpose we studied the
effects of the length of the starvation period on A~b
and ApH. Chemostat cultures were pulsed with a
mixture of [14C]benzoic acid (probe for ApH) and
[3H]Ph4 P+ (probe for A~b). After 5 min incubation the culture was harvested and at various times
samples were withdrawn, quickly filtered and
counted for radioactivity. The entire sampling and
filtration procedure required 10-15 s. Since previous experiments had shown that the magnitude of
the components of the pmf varied significantly
with the external pH we performed our measurements in chemostat cultures which were fixed at
p H 5.7 (pmf is composed of ApH and A~b) and at
p H 7.5 (only Aq~ contributes to the pmf). The
results of the lactose starvation experiments (Fig.
1A,B) show that the membrane potential remained
intact for at least 2 min. Subsequently, the Aft
dropped rapidly and was immeasurably low after
30 rain starvation. The time course of decay of the
ApH followed a completely different pattern.
Lactose starvation for at least 30 min did not
seriously affect the ApH. These results clearly
demonstrate that the components of the p m f in
chemostat-grown cells of S. cremoris can be measured accurately with this assay procedure.
3.1. Effect of growth rate and external p H on the
magnitude and composition of the p m f
The components of the p m f were measured in
S. cremoris growing in lactose-limited chemostat
cultures at various culture pH values and different
dilution rates (Fig. 2). While the ApH component
was hardly affected by the growth rate, A4, increased with decreasing growth rate at all pH
values tested. As a result the p m f increased with
decreasing growth rate. The chemostat experiments also revealed that culture p H had a pronounced effect on the magnitude of both components of the pmf. At the lowest p H tested (pH 5.7)
the ApH was approx. --70 mV at p H 6.4, --40
mV and at p H 7.0, - 2 5 inV. The pH of the
cytoplasm thus lies between 6.7 and 7.0. These
results show that the pH of the cytoplasm of
growing cells is regulated within small limits and
that the value of the cytoplasmic p H is apparently
independent of growth rate. The membrane poten-
pH 5.7
o
A
u pH 6./. , BI
pH 7.0
o
~15(
o
10[
o e oee
01
02
03
01
02
03
•
01
02
0.3
specific growth rote [l/h)
Fig. 2. Effect of growth rate on the composition (0, A6; Q,
ApH) and magnitude of the total pmf (rq) in S. cremoris,
growing lactose-limited chemostat cultures at three different
pH values: (A) pH 5.7; (B) pH 6.4 and (C) pH 7.0. The growth
rate of the culture was varied by changing the dilution rate of
the culture.
tial was also strongly affected by the culture pH
and increased from 80-90 mV (negative inside) at
p H 5.7, to 100-130 mV at pH 7.0. As a result of
these variations of both A4, and A p H the total pmf
remained remarkably constant over the pH range
5.7 7.0.
3.2. Effect of growth rate on the rate of lactose
breakdown
The apparent increase of the p m f at low growth
rates is an interesting phenomenon. Possibly this
increase is the result of an increased rate of ATP
production. Since ATP in S. cremoris is exclusively
produced by substrate level phosphorylation an
increased rate of ATP synthesis would be accompanied by an enhanced rate of lactate production.
The specific rate of lactate production was therefore determined at various growth rates. The
specific rate of lactate production increased linearly with the growth rate at all culture pH tested
(data not shown). Such a relationship can be expected for a culture of S. cremoris which is limited
by lactose [6]. This result clearly shows that the
observed increase of the pmf at low growth rates
cannot be explained by an increased rate of lactose
breakdown to lactate. An alternative explanation
73
might be an increased production of ATP by
substrate level phosphorylation. It is frequently
observed that lactic streptococci change from a
homo- to a heterolactic type of fermentation at
low growth rates [13]. This change in fermentation
pattern is accompanied by an increased production of ATP by substrate level phosphorylation.
Analysis of the fermentation products at several
growth rates, however, showed that S. cremoris
Wg2 converted lactose almost entirely to lactate
(more than 95%) and this fermentation pattern did
not alter at low growth rates.
4. DISCUSSION
The data in this report clearly show that the
pmf of S. cremoris increases at low growth rates.
This observation was unexpected since we considered lactose-limited growth equivalent to energy-limited growth, a condition which we anticipated would result in a direct proportional relationshipbetween the pmf and growth rate.
S. cremoris converts lactose for more than 95%
to lactate and this fraction of lactose serves as
energy source. However, a small but significant
fraction of the available lactose is incorporated
into structural cell material, and thereby serves as
carbon source (R. Otto and R. Lageveen, unpublished results). It is known that fermentable
carbohydrates serve both as energy source and
carbon source for microorganisms growing in very
rich media [14]. We therefore cannot decide
whether growth of S. cremoris in a lactose-limited
chemostat is limited by the availability of energy
or carbon. This aspect remains open for investigation. This point is emphasized since on theoretical
grounds it can be predicted that carbon- or energy-limited growth have a fundamentally different impact on the relationship between the pmf
and growth rate [15]. In contrast to energy-limited
growth, carbon-limited growth would result in an
inversely proportional relationship between the
pmf and growth rate. Our results, therefore, suggest that growth of S. cremoris is limited by the
carbon supply. The apparent increase of the pmf
at low growth rates was the result of an increase of
the membrane potential. The ApH remained fairly
constant which clearly indicates that the cytoplasmic pH is growth-rate independent. The observation that cytoplasmic pH varied only slightly
with external pH is in agreement with the observa 2
tion that the maintenance coefficient of S. cremoris~
increases below pH 7.0 (R. Otto and B. ten Brink~:
unpublished data). Harold [16] suggested that tWo
mechanisms which are simultaneously operative
are responsible for the regulation of the intracellular pH (i) a mechanism responsible for raising the
intracellular pH, as can be accomplished by extrusion of protons via the membrane bound ATPase
combined with electrogenic uptake of potassium;
(ii) a mechanism responsible for lowering the intracellular pH. This can be accomplished by electroneutral proton uptake via a proton-potassium
antiporter or by the electrogenic uptake of protons
via passive diffusion.
Our experiments in which we studied the time
course of A+ decay after breaking off the medium
supply allow an estimation of leak processes during lactose-limited growth. The number of protons
(or other monovalent cations) needed to discharge
the membrane can be calculated with the following
formula:
mol H + - C . V
E.N
(1)
where C represents the electrical capacity of the
cytoplasmic membrane (in farad (F)/m2), V the
membrane potential (volts), E the elementary
charge (1.6.10 -19 coulomb (C)/charge) and N
Avogadro's number (6.02. 10 23 molecules/mol). A
streptococcus culture with a density of 1 mg cell
dry weight/ml contains approx. 0.5-10 I° cells.
Taking a value for the total surface area of a single
streptococcus cell of 3.4- 10- 12 m 2 [17] and for the
electrical capacity of a biological membrane a
value of 10 2 F / m 2 [18] then the total electrical
capacity of 1 mg cells is 1.7 • 10 4F. At pH 7.6 the
A+ is -125 mV. From Fig. 1B it can be calculated
that the maximal rate of A~b dissipation is 7.5
mV/min.-Using Eqn. 1, the amount of protons
needed for dissipating the existing A~p is 1.3 • 10 5
/~mol H + / m i n / m g dry weight (or using a value
for the intracellular water space of 1.8/~l/mg dry
weight: 7.2/~mol H + / m i n / l cell water). This value
is considerably lower (3500-fold) than the value
74
given b y M a l o n e y for the passive i n f l o w of p r o t o n s
i n starved cells of S. lactis [19]. H o w e v e r , it m u s t
b e kept in m i n d that o u r c a l c u l a t i o n s give a n
e s t i m a t e of the net i n f l u x of p r o t o n s . P r e l i m i n a r y
e x p e r i m e n t s with S. cremoris s h o w e d that u p o n
e x h a u s t i o n of lactose the decrease of the A+ coincides with a sharp fall of the i n t r a c e l l u l a r A T P a n d
A D P c o n c e n t r a t i o n s (R. O t t o a n d B. K l o n t , u n p u b l i s h e d results). C l e a r l y the rate at w h i c h the A~b
is d i s s i p a t e d i n S. cremoris is the r e s u l t a n t of
passive H + i n f l o w a n d H + e x t r u s i o n b y the m e m b r a n e - b o u n d Ca 2 + , M g 2 + - s t i m u l a t e d A T P a s e . T h e
true passive i n f l o w of H + d u r i n g g r o w t h of S.
cremoris will therefore b e c o n s i d e r a b l y higher.
ACKNOWLEDGEMENT
T h e i n v e s t i g a t i o n s were s u p p o r t e d b y the
F o u n d a t i o n for F u n d a m e n t a l Biological R e s e a r c h
( B I O N ) w h i c h is s u b s i d i z e d b y the N e t h e r l a n d s
O r g a n i z a t i o n for the A d v a n c e m e n t of Pure Research ( Z W O ) .
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