Previous Article | Next Article 
Journal of Bacteriology, February 2000, p. 1136-1143, Vol. 182, No. 4
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Metabolic Network of Lactococcus
lactis: Distribution of 14C-Labeled Substrates between
Catabolic and Anabolic Pathways
L.
Novák and
P.
Loubiere*
Centre de Bioingénierie Gilbert Durand,
UMR CNRS 5504, UR 792 INRA, Institut National des Sciences
Appliquées, Complexe Scientifique de Rangueil, F-31077 Toulouse
Cedex 4, France
Received 22 April 1999/Accepted 22 November 1999
 |
ABSTRACT |
Lactococcus lactis NCDO 2118 was grown in a simple
synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the
carbon fluxes into the metabolic network. The specific rates of
substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon
fluxes within the catabolic and anabolic pathways. The macromolecular
composition of the biomass was measured to distribute the global
anabolic flux into the specific anabolic pathways. Finally, the
distribution of radiolabeled substrates, both into the excreted
fermentation end products and into the different macromolecular
fractions of biomass, was monitored. The classical end products of
lactic acid metabolism (lactate, formate, and acetate) were labeled
with glucose, which did not label other excreted products, and to a
lesser extent with serine, which was deaminated to pyruvate and
represented approximately 10% of the pyruvate flux. Other minor
products, keto and hydroxy acids, were produced from glutamate and
branched-chain amino acids via deamination and subsequent
decarboxylation and/or reduction. Glucose labeled all biomass fractions
and accounted for 66% of the cellular carbon, although this
represented only 5% of the consumed glucose.
| |
INTRODUCTION |
Lactic acid bacteria (LAB) are of
great importance in the food industry, mainly for lactic acid
production from various substrates, but also for flavor compound or
bacteriocin synthesis. The nutritional complexity of LAB is such that
they are frequently cultivated in media containing complex nitrogen
sources (MRS [9] or M17 [30]) or in
natural media (milk or wine) for different applications. However, the
complexity of these media is such that growth and metabolic behavior
are difficult to characterize precisely. This is certainly the reason
why it is generally considered that during LAB fermentation, sugar is
only a catabolic substrate leading to metabolic end products and energy
while biomass is formed from anabolic precursors, i.e., amino acids,
nucleotides, etc., present in the culture broth. Whatever the medium
used, more than 90% of the carbon in the sugar is normally converted
into metabolic end products, generally, lactic acid. Moreover, the
growth of LAB is characterized by poor growth yield, i.e., amount of
biomass formed per amount sugar consumed, and hence, the quantity of
the biomass formed is low compared to the quantity of lactic acid produced. However, this simplistic model is based on inaccurate carbon
balances and is very controversial. A very small part of the sugar
leading to anabolic reactions could represent a significant part of the
biomass carbon, and this consideration has been totally neglected.
The study of radiolabeled substrate distribution into end products or
biomass has been used to estimate the part of sugar leading to biomass.
Sivakanesan and Dawes (29) reported that 0.5% of the
glucose used as the substrate labeled the biomass of
Staphylococcus epidermidis. Chauvet et al. (6)
cultivated Leuconostoc oenos in wine supplemented with
labeled substrates, e.g., glucose and malic and citric acids. They
demonstrated that 2.1% of the glucose was recovered as biomass, while
neither malic acid nor citric acid was a precursor of anabolic
compounds. Later, Schmitt et al. (27) showed that during the
cultivation of L. mesenteroides subsp. cremoris
in complex MRS medium, [14C]glucose was not incorporated
into biomass, while 1.1% of the citrate present was incorporated into
biomass in the form of acetyl coenzyme A (acetyl-CoA). Benthin et al.
(2) suggested that in steady-state and transient cultures of
Lactococcus cremoris FD1 in a chemostat, a small part of the
carbon in the sugar might contribute to biomass formation. Plihon et
al. (22) performed batch experiments with L. mesenteroides in MRS medium, and they concluded that "the carbon
balance confirmed that biomass was not created from sugar
metabolism." Clearly, this conclusion is subject to doubt since the
experimental error observed in the substrate and product concentrations
given by high-pressure liquid chromatography (HPLC) was 5%,
illustrating that this kind of experiment is unable to give precise
information about the precursors of biomass synthesis.
On the other hand, some evidence exists to support the idea that
certain amino acids can be catabolized and metabolic products can be
excreted into the culture medium. This is established for some amino
acids converted to oxo or hydroxy acids involved in cheese flavor
compounds (36). It has also been shown that serine can be
deaminated to give pyruvate and then lactate (3, 18). In
this case, the operation of this catabolic pathway modifies the
calculated value of the lactate yield from the sugar and confirms that
carbon balancing based solely upon sugar-to-product conversion cannot
be used alone to draw conclusions about carbon flux distribution.
A precise study of carbon distribution into biomass requires that the
macromolecular composition of the cells be known. The data previously
published for the composition of L. lactis are difficult to
generalize because they were obtained with a variety of strains and
with various methods and, furthermore, only certain biomass components
were assessed. Moreover, both growth conditions and growth rate can
influence the percentage of each class of macromolecules or the
chemical composition of a macromolecular fraction (5, 34).
For example, the lipidic composition of Escherichia coli is
affected by the pH, temperature, aeration, or growth rate
(1). The macromolecular composition of L. lactis NCDO 712 is also dependent on the growth rate, particularly with regard
to RNA/protein or RNA/DNA content ratios (4). Significant variation of protein content has been reported: Thomas and Batt (31) showed that L. lactis ML3 contains 48%
protein, including the peptides of peptidoglycan, while Benthin et al.
(2) reported a value of 45% for L. cremoris, not
including the peptides of the cell wall. A very different value,
between 23 and 30%, was obtained by Foucaud (10) for
L. lactis subsp. lactis biovar diacetylactis CNRZ
125 or 141.
To date, no data have been reported on the detailed metabolic network
of L. lactis, since these data necessitate quantitative determination of macromolecular biomass composition, precise
determination of fermentation kinetics, and measurement of the
distribution of 14C-labeled substrates into the cell,
performed together with one strain and under one precise set of culture
conditions. In this study, these various approaches have been combined
to calculate the most complete carbon flux distribution pattern within
the metabolic network of L. lactis NCDO 2118.
| |
MATERIALS AND METHODS |
Organism and culture media.
L. lactis subsp.
lactis NCDO 2118, obtained from the collection held at the
Institut National de la Recherche Agronomique (Jouy-en-Josas, France),
was used throughout this study. The medium used for the growth of the
inoculum was the synthetic MS10 medium described by Cocaign-Bousquet et
al. (7), while the medium used for the experiments was the
MCD medium described by Otto et al. (19) and modified by
Poolman and Konings (23) or MS14 medium (18).
These media were prepared from concentrated stock solutions stored at
4°C after filtration through cellulose nitrate membranes (0.22-µm
pore size), except for the cysteine solution, which was freshly
prepared. The media (pH 6.6) were sterilized by filtration through
cellulose acetate membranes (0.22-µm pore size; Sartorius) directly
into a sterilized (20 min at 121°C) culture vessel.
Culture conditions.
Fermentations were carried out under
strictly anaerobic conditions in a 2-liter glass fermentor
(Sétric Génie Industriel, Toulouse, France) or in anaerobic
tubes at a temperature of 30°C and an agitation speed of 250 rpm. The
bacteria were grown in a controlled gas environment created by flushing
of both the vessel and the medium with nitrogen. The medium in the
fermentor was aseptically gassed (30 min) immediately before
inoculation and maintained under an N2 atmosphere at a
positive pressure of 103 Pa. Fermentor cultures were
maintained at pH 6.6 by automatic addition of 5 N KOH.
Inoculation was at 2% with a high-optical-density (optical density at
580 nm, 2.0) culture on MS10 medium with a high concentration of
phosphates ([K2HPO4 = 15 g/liter and
[KH2PO4] = 18 g/liter) to buffer acid
production, and the culture was incubated overnight at 30°C in a
butyl rubber-stoppered 250-ml shaken flask under N2.
Precultures were washed twice with sterile phosphate buffer (100 mM, pH
6.6) to avoid carryover of essential nutrients and resuspended in the
same buffer. All tube cultures were carried out in triplicate and
repeated if experimental variation exceeded 5%.
Analytical methods.
Bacterial growth was monitored by
spectrophotometric measurements at 580 nm and calibrated against cell
dry weight measurements. Cells were harvested by filtration on
0.45-µm-pore-size nylon membranes, washed with 2 volumes of deionized
water, and dried to a constant weight at 60°C under a partial vacuum
(200 mm Hg [ca. 26.7 kPa]). A change of 1 U of optical density was
shown to be equivalent to 0.31 g of dry matter · liter
1. The biomass formula was determined at ENSC
(Toulouse, France) by elemental analysis of C, H, O, N, and ash. The
ash fraction on MS14 medium corresponded to 9.2% (wt/wt) of the
biomass. The biomass formula used to convert cell dry weights into
molar cell carbon concentrations was
C5.05H9.20O2.77N1.0,
with a molar mass of 140 g · mol1, including ash
at 12 g · mol1.
The carbon dioxide concentration in the gas phase was determined by gas
chromatography using a Porapak Q column followed by
a molecular sieve
maintained at 40°C with helium as the carrier
gas and catharometer
detection.
Determination of sugars and organic acids from fermentation
supernatants or from cell hydrolysates was performed by HPLC using
an
Aminex HPX-87H
+ column (300 by 7.8 mm) and the following
conditions: a temperature
of 50°C, solvent
H
2SO
4 (5 mM), a flow rate of 0.5 ml · min
1, and dual detection (refractometer and UV at 220 nm). Ethanol
concentration measurements were also carried out with
isocratic
gas chromatography on a Porapak-Q packed column at 190°C
with
a flame ionization detector (Intersmat GC). Some compounds
produced
during the fermentation and visualized by peaks during HPLC
analysis
were identified by checking the retention time with standard
solutions.
Ammonia concentrations were determined in filtered culture samples with
an ammonia-selective electrode
(Orion).
Concentrations of amino acids in the medium were determined with an
AminoQuant 1090 HPLC apparatus (Hewlett-Packard) after
derivatization
by
ortho-phtalaldehyde in the presence of
3-mercaptopropionic
acid and by 9-fluorenylmethoxy carbonyl, separation
with a C
18 column, and spectrophotometric detection at 338 nm for
ortho-phtalaldehyde
derivatives or 266 nm for
9-fluorenylmethoxy carbonyl
derivatives.
For the separation of radiolabeled amino acids, micellar HPLC on a
Beckman Ultrasphere octyldecyl silane column (250 by 4.6
mm) was done
by using a modification of the method of Saurina
and Hernandez-Cassou
(
25). Eluent A contained H
3PO
4 (20 mM),
Na
2HPO
4 (20 mM), and sodium dodecyl
sulfate (10 mM) in deionized
(MilliQ) water. In eluent B, water was
replaced with a mixture
of deionized water-acetonitrile (60/40). The
flow rate was maintained
constant at 0.15 ml/min throughout the entire
chromatographic
run, while the temperature was kept at 50°C.
Separation of amino
acids was performed by an initial isocratic elution
(100% eluent
A from 0 to 80 min), followed by a linear gradient from
80 min
(0% B) to 560 min (100% B). The percentage of eluent B was
linearly
decreased afterward and then maintained at 0% until
re-equilibration
of the column. Detection of amino acids was performed
by measurement
of UV absorption at 200 nm, while in-flux radioactive
countings
were done as described
below.
Determination of biomass macromolecular composition.
The
cells were centrifuged (4°C, 10 min at 8,000 × g),
washed three times with MgCl2 (1 to 5 mM), and then
lyophilized. These cells were used for the quantification of each
macromolecular biomass fraction.
Carbohydrates were measured by three methods, two colorimetric methods
with anthrone or phenol (
14), and one chromatographic
method. For this last method, lyophilized cells were resuspended
in
distilled water, sonicated to disrupt the cell wall, and sealed
in a
tube under nitrogen with 25 to 50 mg of Dowex 50W×4 (200
to 400 mesh)
resin. Hydrolysis was done at 115 to 125°C for 3
to 12 h. Sugars
were separated by HPLC on an Aminex HPX-87H
+ column (see
the description of analytical
methods).
After cell wall disruption by sonic treatment, lipidic compounds were
extracted with
n-hexane-isopropanol (3:2) as described
by
Hara and Radin (
13) and weighed after
drying.
Fatty acids were measured in cells incubated (15 min, 80°C) in
concentrated HCl under argon. Methanol was added to this suspension
and
then esterified under argon (2 h, 80°C). Fatty acid esters
were
extracted with hexane, dried under argon flux, and recovered
in a few
milliliters of hexane. This extract was analyzed by gas
chromatography
(Hewlett-Packard 5890 Series II) by injection into
a capillary column
(Supelco SP2330, 30 m by 0.25 mm) at 200°C
with nitrogen as the
carrier
gas.
Amino sugars constitutive of the peptidoglycan were measured after
hydrolysis of the cell wall. Lyophilized cells were incubated
(2 to
6 h, 105°C) in a sealed tube containing HCl (4 M) and under
nitrogen. The hydrolysate was evaporated under vacuum, resuspended
in
distilled water, and passed through a Dowex 50W×4 column. The
column
was washed with distilled water and eluted with HCl (2
M), and the
eluate was dried under vacuum. Amino sugars were measured
spectrophotometrically after reaction with dimethylaminobenzaldehyde
(
11,
12) or by
HPLC.
Nucleic acids were extracted from lyophilized cells by incubation with
perchloric acid (0.5 M, 70 to 80°C, 15 to 20 min) and
measured as
described by Hanson and Phillips (
12).
After solubilization of the cells with NaOH (1 M, 90°C, 10 min)
proteins were determined by the biuret method, the Folin reagent,
or
the use of Coomassie blue (
21). The amino acid content of
the proteins was determined after hydrolysis (105°C, 24 to 36
h)
under nitrogen by a modified form of the method of Ng et al.
(
17), the hydrolyzing solution containing 2-mercaptoethanol
(0.2 or 1%), or a modified form of the method of Yano et al.
(
35)
with a mixture of chlorhydric (7 M), trifluoroacetic
(10%), and
thioglycolic (20%) acids and phenol (1%). Lysozyme was
used as
a standard solution to quantify the loss of amino acids during
hydrolysis.
Use of radiolabeled substrates.
All of the uniformly
14C-radiolabeled substrates were obtained from Amersham and
used at 8 to 50 kBq/ml of medium.
Measurement of radioactivity in glucose and in the fermentation end
products was done by passing the sample through an HPLC
column (see the
description of analytical methods), followed by
detection with a
continuous scintillation counter (Berthold 506A).
The scintillation
fluid was mixed with the HPLC eluant at a ratio
of 2:1 and counted in
the
detector.
Radioactivity in the biomass macromolecules was determined by using
cells washed three times with MgCl
2 (5 mM) and counted
in
each fraction obtained after sequential fractionation of the
biomass as
described previously by Paalme et al. (
20).
Balance and flux calculations.
The total carbon balance was
calculated as the amount of the products and biomass formed during the
entire duration of the culture period as a function of the
concentration of glucose and amino acids consumed. The apparent
catabolic balance was calculated as the ratio of the products formed,
i.e., lactate, formate, acetate, and pyruvate, to the glucose consumed.
The specific rates of substrate consumption or product formation,
expressed in millimoles per gram per hour, were calculated
from the
kinetic data of the substrate and product concentrations
measured
during the culture period. All of the specific rate values
were
maintained constant during the major part of the culture
at their
maximal value. These maximal values were used to calculate
the fluxes
through the metabolic pathways and correspond to the
molecular fluxes
at the input and output of the metabolic pathways.
The conceptual basis
and mathematical expression for such flux
calculation have been
described by Vallino and Stephanopoulos
(
32) and used
previously for other bacteria (
8). The metabolic
fluxes were
better expressed as carbon flux by taking into account
the number of
carbon atoms in each molecule. The biomass can be
considered a
macromolecule with molar mass
M (in grams per mole)
and
macroelemental composition
C
aH
bO
cN
d which is
formed at specific
growth rate µ (per hour). The flux of synthesis of
a biomass macromolecule
is the product of its concentration in the
biomass (in millimoles
per gram) and the growth rate, µ.
Calculation of carbon substrate incorporation into the carbon of a
biomass macromolecular fraction was done with the equation
I =
Af ·
Mx ·
As1 (
ns/
nx),
where
I is the micromoles of substrate carbon incorporated
by the micromoles of biomass carbon,
Af is the
specific radioactivity
of fraction
f (in becquerels per
milligram of biomass),
As is
the specific
radioactivity of the substrate (in becquerels per
micromole),
Mx is the molar mass of the biomass (140 g
· mol
1 in MS14 medium),
ns is
the number of carbon atoms in the substrate,
and
nx is the number of carbon atoms in a biomass
molecule (5.05
in MS14 medium). These incorporation values were then
converted
to relative incorporation rates expressed as a function of
the
sum of the values obtained for the different
fractions.
| |
RESULTS AND DISCUSSION |
Batch culture of L. lactis NCDO 2118 in MS14
medium.
The growth and metabolic behavior of L. lactis
NCDO 2118 in MCD or MS14 medium were as previously described (15,
18). The metabolism was homolactic in both media and was
characterized by a lactate yield of about 1.7 mol/mol of glucose. The
identified minor fermentation products were formate, acetate, and
ethanol in MCD medium, while in MS14 medium, pyruvate and ammonium were also excreted into the culture broth and no ethanol was produced (data
not shown). Amino acid consumption was related to the amino acid
composition of the medium. As previously described, large amounts of
serine were consumed in the MS14 medium and associated with ammonia
production (18).
Growth started immediately after inoculation and continued until
glucose had been completely exhausted from the medium. Growth
was more
rapid in MCD medium, with a maximal specific rate of
0.8 h
1 instead of the 0.18 h
1 measured in MS14
medium. In this last medium, the maximal specific
growth rate was the
result of the absence of a number of amino
acids from the medium
(
18).
The specific rates of growth, consumption of both sugar and amino
acids, and product formation rapidly reached their maximal
and were
maintained constant for the major part of the culture
period. These
maximum specific rates, calculated during the exponential
growth phase
in MS14 medium, were as follows (expressed in millimoles
per gram per
hour): glucose consumption, 13.6; glutamate consumption,
0.3; serine
consumption, 3.4; methionine consumption, 0.06; leucine
consumption,
0.4; isoleucine consumption, 0.3; valine consumption,
0.2; lactate
production, 24.2; formate production, 2.4; acetate
production, 2.2;
pyruvate production, 0.4, biomass production,
1.3 (corresponding to a
growth rate of 0.18 g · g
1 · h
1).
The apparent catabolic balance, calculated by the ratio of lactate,
formate, acetate, and pyruvate production to glucose consumption,
showed 96% carbon recovery and might indicate that glucose was
only
catabolized for energy production, as currently assumed.
However, since
serine was massively deaminated to pyruvate and
then reduced to lactate
(
18), this simple balance introduces
flux errors. This
consideration was confirmed by looking at the
total carbon balance,
i.e., products and biomass production from
glucose and the consumed
amino acids, which indicated that only
86% of the consumed carbon was
recovered as biomass carbon or
classical end products of lactic
metabolism. It can therefore
be assumed that other catabolic end
products were formed from
both glucose and amino
acids.
Macromolecular composition of biomass.
The cells cultivated in
MCD or MS14 medium were analyzed to determine the macromolecular
composition of the biomass (Table 1). The
polysaccharide fraction was greater in MS14 medium than in MCD medium,
while the opposite was true of the RNA fraction. Other macromolecular
fractions were identical in cells grown on either of the two media. The
protein fraction, which represents the cellular proteins and the cell
wall peptides, was by far the most important in the cell, with all
other fractions representing between 3 and 15% of the biomass.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Macromolecular composition and amino acid composition of
the protein fraction of L. lactis NCDO 2118 grown in MCD or
MS14 mediuma
|
|
The values obtained for the polysaccharide fraction differed with the
method used. In MCD medium, the anthrone method gave
7.1% while the
phenol method led to 15.3%. A similar difference
was obtained with
MS14 medium, i.e., 9.4 and 17%, respectively.
This difference was due
to the insensitivity of anthrone to deoxyhexoses
(rhamnose) and
pentoses and to the reactivity of phenol with nucleotidic
sugars. In
view of this, the polysaccharide content of the cell
was calculated by
taking into account an estimation of the different
sugars constituting
this fraction. As a consequence, the values
presented in Table
1 are
not arithmetic averages of these two
experimental values but corrected
values. An apparent difference
was observed between our polysaccharide
content values and the
values determined by Thomas and Batt
(
31) for
L. lactis ML3
grown in a complex medium,
but the 7.7% they found was obtained
with anthrone and was similar to
our value of 7.1% obtained with
the same method during growth in MCD
medium. Chromatographic analysis
of this fraction has revealed that
glucose and rhamnose are the
predominant sugars while the cells have a
lower galactose content,
with a respective mass proportion of
5.5/5.1/1.0.
While the level of DNA measured was similar to that observed by Thomas
and Batt (
31), the RNA concentration was lower (6
to 8%
instead of 21%). It is known that the RNA level of the cell
is
susceptible to variation with the metabolic activity and growth
rate of
the cell. This is probably the reason for the high RNA
level observed
by Thomas and Batt (
31), since in their experiments,
growth
proceeded rapidly in a complex medium. Moreover, it has
been previously
observed for
Saccharomyces cerevisiae (
28) and
Klebsiella aerogenes (
16) that growth under
nitrogen-limiting
conditions, as would be the case in the media
employed in this
study, leads to a low RNA
concentration.
The fatty acid composition of the cells determined by HPLC analysis
showed that palmitic and oleic acids (C
16:0 and
C
18:1,
respectively) were present in similar amounts in the
MCD medium
used, while the oleic acid concentration was very low in the
MS14
medium used. Hence, while the lipid concentrations were identical
in the two media tested, and comparable to the data of Thomas
and Batt
(
31), the composition differed with the medium. Moreover,
lactobacillic acid (cyclopropane acid [C
19]) was not
identified
in these cells, whatever the medium used, while it was
detected
in the type species NCDO 712 described by Schleifer et al.
(
26).
The protein fraction of our strain was in good agreement with the
values generally reported. The amino acid content of proteins
was
determined after acidic hydrolysis of the total proteins of
cells
cultivated in MCD or MS14 medium. The different hydrolytic
methods used
gave very similar results for the majority of the
amino acids. Only
tryptophan and cysteine were not detected when
hydrolysis was done with
HCl and 3-mercaptopropionic acid (1%).
The average percentage of each
amino acid in cells cultivated
in either MCD or MS14 medium is shown in
Table
1. It appears
that this amino acid composition was very conserved
when the medium
composition was varied, since the maximal difference
between the
two media used was only 1.5%, as observed for glutamate
plus glutamine
or for tryptophan. Alanine, aspartate-asparagine,
glutamate-glutamine,
lysine, leucine, and glycine were the predominant
amino acids,
each accounting for more than 7% of the protein fraction.
The
predominance of alanine, aspartate, glutamate, and lysine in the
protein fraction was not surprising, since they are constituents
of the
peptidoglycan of
L. lactis (
26). On the other
hand, tryptophan,
histidine, and methionine are the amino acids least
abundant in
the
cell.
Distribution of 14C-labeled substrates in fermentation
end products.
Among the carbon substrates present in MS14 medium,
glucose and five of the six amino acids (glutamate, serine, isoleucine, leucine, and valine) were used separately as uniformly
14C-labeled substrates during the cultures, and the
radioactivity was measured in the fermentation end products. Only
methionine was not tested because it was consumed in very low amounts
and in proportion to the amount of methionine found in the cells.
The HPLC profile obtained with a refractometer detector showed many
peaks, some of them corresponding to glucose and the classical
end
products of lactic fermentation but others being unidentified
(Fig.
1), as classically observed for LAB
cultures. From the radioactivity
measurements, it appeared that all of
the substrates tested led
to the production of excreted compounds.
About 91% of the lactate,
formate, acetate, and pyruvate found came
from glucose, which
did not give other products. The other 9% of these
products derived
from pyruvate, originating from serine. Hence, the
classical products
of lactic fermentation were derived specifically
from glucose
and serine.
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 1.
Origin of fermentation end products of L. lactis NCDO 2118 grown in MS14 medium, determined by HPLC
analysis, followed by radioactivity measurement. In the graphs at the
top, the HPLC refractometric signal is plotted, showing all of the
detected compounds, before (left) and after (right) glucose
exhaustion. In the other graphs, the radioactivity signal after culture
with various 14C-labeled substrates (written on the graph)
is plotted, showing the compounds derived from the corresponding
labeled substrates. Peaks: 1, phosphate; 2, 2-ketoglutarate; 3, pyruvate; 4, glucose; 5, 3-hydroxyisobutyrate; 6, 3-hydroxymethylbutyrate; 7, 3-hydroxyisovalerate; 8, lactate; 9, formate; 10, acetate; 11, 2-hydroxyisovalerate; 12, pyroglutamate; 13, 2-hydroxyisocaproate; 14, 2-hydroxymethylvalerate. RI, refractive
index.
|
|
Glutamate gave two particular compounds produced in low concentrations,
2-ketoglutarate and pyroglutamate, a product of cyclization
by
dehydration between amine and
-carboxylic groups of the
molecule.
Each branched-chain amino acid produced two different compounds, some
of them leading to important peaks observed by refractometry
(peaks 13 and 14). None of these compounds were the keto acids
produced by
deamination (or transamination) of the amino acids
(for example,
2-ketoisocaproate from leucine). However, the 2-hydroxy
acids derived
from each of these keto acids by a reduction step,
e.g.,
2-hydroxyisocaproate from leucine, 2-hydroxy-3-methylvalerate
from
isoleucine, and 2-hydroxyisovalerate from valine, were present
in the
medium. These hydroxy acids were identified by reducing
in vitro the
respective keto acids by sodium borohydride in phosphate
buffer (pH
7.2) and checking the retention times of these compounds
in HPLC
(
33). Moreover, this reduction was also achieved in
vitro by
lactate dehydrogenase in a phosphate buffer (pH 7.2)
containing NADH.
In this assay, not only did NADH absorption gradually
decrease but
hydroxy acids also appeared in the cuvette. The three
other peaks
observed from the three branched-chain amino acids
(Fig.
1, peaks 5, 6, and 7) should correspond, from retention
time data, to the 3-hydroxy
acids obtained after successive deamination,
decarboxylation, and
oxidation of each amino acid. In conclusion,
all amino acids not
directly included in the biomass were deaminated
into keto acids, a
part of them being reduced with NADH, and this
reduction could be
catalyzed by lactate
dehydrogenase.
Distribution of 14C-labeled substrates in biomass
carbon.
Cells cultivated in MS14 medium with
14C-labeled substrates were harvested during the
exponential growth phase and fractionated by a sequential procedure to
obtain the various macromolecular fractions. The radioactivity present
in each fraction was then counted and expressed as the percentage of
carbon from the labeled substrate recovered in each fraction (Table
2). The macromolecular fractions obtained
after sequential fractionation of the biomass differed from the
fractions recovered by macromolecular analysis, since complete
separation by the sequential method was impossible. This was
particularly true for the last separation steps. Care should be taken
in analyzing these results, since low radiolabeling values could be due
to contamination of a fraction with other molecules. Nevertheless, this
method offers the advantage of simplicity and gives results
complementary to the analysis of biomass composition.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Distribution of 14C-labeled substrates
(glucose and amino acids of MS14 medium) in biomass fractions obtained
by sequential fractionationa
|
|
The pool fraction represented 4.1% of the total biomass carbon. This
fraction was issued for 92% of the molecules originating
from two
substrates, glucose (51.3%) and glutamate (40.4%). The
other four
substrates accounted for only 8.2% of the pool fraction.
The very high
concentration of glutamate or glutamate-derived
products in the cytosol
is in agreement with the previous observations
of Thomas and Batt
(
31) for
L. lactis ML3 or of Poolman et al.
(
24) for
L. lactis subsp.
cremoris
Wg2. Glucose gave 84.6% of
the lipidic fraction, glutamate gave 5.4%,
and serine gave 6.3%,
while the three branched-chain amino acids
labeled no more than
4.0%. The fraction containing RNA and a part of
teichoic acid
was labeled predominantly by glucose (87.4%), and a
small part
was labeled by serine (5.4%), while the other four
substrates
labeled only 7.2%. The following fraction, containing DNA,
polysaccharides,
and also some peptides, was labeled as follows:
glucose, 76.2%;
glutamate, 8.4%; serine, 4.6%; valine, 4.3%;
isoleucine, 3.6%;
leucine, 2.9%. Finally, the protein fraction was
labeled as follows:
49% by glucose, 19.7% by glutamate, 5.9% by
serine, 7.6% by valine,
7.5% by isoleucine, and 10.4% by leucine.
These results are coherent
with the knowledge of biochemical pathways
and the metabolic precursors
of each class of macromolecules. Lipids
originated predominantly
from glucose via acetyl-CoA, RNA originated
from glucose via the
pentose pathway for the ribose moiety and via the
tricarboxylic
acid cycle and the serine family for the nucleotides, and
teichoic
acid originated from glucose via glycerol phosphate,
galactose,
and alanine (
26). The large percentage of the DNA
fraction and
the presence of a significant amount of label from each
amino
acid indicate that important polysaccharide and soluble peptide
cross-contamination might contribute to this
fraction.
In MS14 medium, glucose was the predominant precursor of each
macromolecular fraction and gave 66% of the total carbon biomass.
The
same experiment was realized in MCD medium with a complete
amino acid
composition and nucleotides with [
14C]glucose as the
substrate. In this case, glucose labeled 33%
of the total carbon
biomass (data not
shown).
Since the protein fraction harvested from cells cultivated in MS14
medium was highly labeled by glucose, the distribution
of radioactivity
from each substrate to the amino acids of cellular
proteins was
determined (Table
3). Glucose gave the
totality
of the amino acids of the phosphoribosyl pyrophosphate pathway
(His, Tyr, and Phe), about 90% of the alanine-and-aspartate family
(Asp, Asn, Thr, and Lys), a small fraction of arginine, and, curiously,
the majority of the glycine. Data for tryptophan were not available,
since it was destroyed during protein hydrolysis, but it can be
assumed
that it was produced from glucose, as were Tyr and Phe,
and also from
serine. Glutamate labeled only the amino acids of
its own family (Glu,
Gln, Pro, and Arg). Serine labeled the amino
acids coming from pyruvate
(Ala) and the tricarboxylic acid cycle
(Asp, Asn, Thr, and Lys) in a
proportion of about 6 to 10%. Glycine,
which belongs to the serine
family, was, however, labeled at only
33% by serine. The
branched-chain amino acids of the biomass originated
directly from
those present in the medium. Moreover, Ile and Leu
labeled Arg and the
amino acids of the aspartate family at a very
low level.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Distribution of 14C-labeled substrates of
MS14 medium in the amino acids of cellular proteins of L. lactis NCDO 2118
|
|
Metabolic network of L. lactis NCDO 2118 in MS14
medium.
Data on 14C distribution into the
macromolecular fractions enable the global metabolic scheme operating
in this medium to be drawn. Taking into account kinetic values of flux
of substrate consumption, biomass and product formation, and the
relative quantity of each macromolecular fraction of the biomass has
enabled the carbon flux through each metabolic pathway to be estimated
(Fig. 2).
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 2.
Carbon fluxes, expressed in micromoles of carbon per
gram per hour, operating during growth of L. lactis NCDO
2118 in minimal synthetic medium MS14. Carbon substrates present in the
culture medium are in boldface roman type, identified excreted products
are in boldface italic type, supposed excreted products are in
lightface italic type, and dashed arrows show possible pathways which
could lead to glycine formation (maximum and minimum carbon flux values
are given for the concerned products, depending on the operative
pathway for glycine synthesis). P, phosphate; diP, diphosphate; PRPP,
phosphoribosyl pyrophosphate.
|
|
The conclusions deduced from carbon balance estimations were confirmed
by the study of distribution of
14C-labeled substrates in
the metabolic network. The major catabolic
process is the degradation
of glucose by the glycolytic pathway.
The specific rate of glucose
consumption calculated during the
exponential growth phase (13.6 mmol
of glucose · g
1 · h
1)
corresponded to a carbon flux input entering glycolysis of 81,400
µmol of C · g
1 · h
1 (Fig.
2). As shown in Tables
2 and
3, a part of the carbon
derived from
glucose and catabolized by glycolysis was used to
give the totality of
polysaccharides, amino sugars, phospholipids,
glycerol, histidine, and
aromatic amino acid fractions and a significant
part of nucleotides,
glycine, alanine, and the aspartate family.
The sum of these carbon
outputs toward biomass synthesis from
glycolysis represented 8% of the
initial glucose carbon input.
Therefore, 92% of the initial carbon
flux will pass through the
entire glycolysis process to reach pyruvate.
Another important
carbon flux arises from serine at the level of
pyruvate (72% of
the initial serine flux) to give metabolites
originating from
pyruvate. The pyruvate flux coming from serine
represents 10%
of that originating from glucose, and the same
proportion is observed
at the level of lactate synthesis. Only 33% of
the glycine came
from serine, despite an apparently functional serine
demethylation
step via tetrahydrofolate. The pathway of glycine
synthesis from
glucose cannot be predicted from these data, since
different possibilities
could be operative. Glycine could be formed
from threonine by
threonine aldolase or 2-amino-3-oxobutyrate synthase
or by transamination
of glyoxylic acid, which could be produced by
isocitrate lyase
or from oxaloacetate via oxalate and oxalyl-CoA.
Isocitrate lyase
activity could not be detected in previous studies
with this strain
(
15), but complementary studies are
necessary to determine the
nature of glycine synthesis in
L. lactis.
Glutamic acid metabolism is distinct from the other metabolic pathways,
because it gives only amino acids of its own family
and two products,
including ketoglutarate. Moreover, this last
metabolite was not labeled
by glucose, indicating that the tricarboxylic
acid cycle was not
operative in this medium, while it was shown
to be functional, although
at a low rate, in a similar synthetic
medium lacking glutamate
(
15).
Each branched-chain amino acid contributed to biomass formation by
itself in the protein fraction but also by participating
in the
carboxylation reactions operating in the biosynthesis of
some
metabolites (pyrimidines, Asp, Thr, Lys, and Arg), since
CO
2 was produced during decarboxylation of the
corresponding keto
acids. Pentose sugar formation from glucose and
branched-chain
amino acid metabolism under anaerobic conditions are the
major
CO
2-generating
pathways.
Despite the complex analysis of the metabolic network, some products
originating from each consumed substrate remain unidentified.
They
represent 13.5% for isoleucine to 25.5% for serine, which
can be
considered low values relative to the total carbon fluxes
but can be of
industrial importance, since some pathways probably
lead to flavoring
compounds of interest in milk
transformation.
Finally, 66% of the carbon biomass produced in this simple synthetic
medium originating from glucose represented only 5% of
the glucose
carbon consumed, explaining the current assumption
that glucose is only
a catabolic substrate. The absence of glucose
incorporation into
biomass reported by Schmitt et al. (
27) might
be due to the
consumption of other carbohydrates present in the
complex medium they
used. The part of glucose incorporated into
biomass was reduced twofold
in MCD medium containing all of the
amino acids and nucleotides, in
good agreement with the modified
anabolic pathways operating in such a
medium, for which synthesis
of anabolic precursors present in the
medium would no longer be
necessary.
| |
ACKNOWLEDGMENTS |
We thank Muriel Cocaign-Bousquet, Nic Lindley, and Attila
Szentirmai for useful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSA,
Département de Génie Biochimique et Alimentaire, 135 Ave.
de Rangueil, F-31077 Toulouse Cedex 4, France. Phone: (33) 5 61 55 94 38. Fax: (33) 5 61 55 94 02. E-mail: loubiere{at}insa-tlse.fr.
| |
REFERENCES |
| 1.
|
Arneborg, N.,
A. S. Salskov-Iversen, and T. E. Mathiasen.
1993.
The effect of growth rate and other growth conditions on the lipid composition of Escherichia coli.
Appl. Microbiol. Biotechnol.
39:353-357.
|
| 2.
|
Benthin, S.,
U. Schulze,
J. Nielsen, and J. Villadsen.
1994.
Growth energetics of Lactococcus cremoris FD1 during energy-, carbon-, and nitrogen-limitation in steady state and transient cultures.
Chem. Eng. Sci.
49:589-609[CrossRef].
|
| 3.
|
Benthin, S., and J. Villadsen.
1996.
Amino acid utilization by Lactococcus lactis subsp. cremoris FD1 during growth on yeast or casein peptone.
J. Appl. Microbiol.
80:65-72.
|
| 4.
|
Beresford, T., and S. Condon.
1993.
Physiological and genetic regulation of rRNA synthesis in Lactococcus.
J. Gen. Microbiol.
139:2009-2017[Abstract/Free Full Text].
|
| 5.
|
Bremer, H., and P. P. Dennis.
1987.
Modulation of chemical composition and other parameters of the cell by growth rate, p. 1527-1542.
In
F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C.
|
| 6.
|
Chauvet, J.,
P. Brechot,
C. Dubois,
P. Dupuy, and J.-L. Dorange.
1982.
Stimulation de la croissance dans le vin d'une flore malolactique par les acides malique et citrique.
Sci. Aliment.
2:495-504.
|
| 7.
|
Cocaign-Bousquet, M.,
C. Garrigues,
L. Novák,
N. D. Lindley, and P. Loubière.
1995.
Rational development of a simple synthetic medium for the sustained growth of Lactococcus lactis.
J. Appl. Bacteriol.
79:108-116.
|
| 8.
|
Cocaign-Bousquet, M., and N. D. Lindley.
1995.
Pyruvate overflow and carbon flux within the central metabolic pathways of Corynebacterium glutamicum during growth on lactate.
Enzyme Microb. Technol.
17:260-267[CrossRef].
|
| 9.
|
de Man, J. C.,
M. Rogosa, and M. E. Sharpe.
1960.
A medium for the cultivation of lactobacilli.
J. Appl. Bacteriol.
23:130-135.
|
| 10.
|
Foucaud, C.
1990.
Physiologie de Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 125 en conditions non optimales. Ph.D. thesis.
Université Claude Bernard Lyon I, Lyon, France.
|
| 11.
|
Ghuysen, J. M.,
D. J. Tipper, and J. L. Strominger.
1966.
Enzymes that degrade bacterial cell walls. Determination of total hexosamines.
Methods Enzymol.
8:692-693.
|
| 12.
|
Hanson, R. S., and J. A. Phillips.
1981.
Chemical composition, p. 335-337.
In
P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Kroeg, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C.
|
| 13.
|
Hara, A., and N. S. Radin.
1978.
Lipid extraction of tissues with a low-toxicity solvent.
Anal. Biochem.
90:420-426[CrossRef][Medline].
|
| 14.
|
Herbert, D.,
P. J. Phipps, and R. E. Strange.
1971.
Chemical analysis of microbial cells.
Methods Microbiol.
5B:265-278.
|
| 15.
|
Lapujade, P.,
M. Cocaign-Bousquet, and P. Loubière.
1998.
Glutamate biosynthesis in Lactococcus lactis subsp. lactis NCDO 2118.
Appl. Environ. Microbiol.
64:2485-2489[Abstract/Free Full Text].
|
| 16.
|
Mulder, M. M.,
H. M. L. Van Der Gulden,
P. W. Postma, and K. Van Dam.
1988.
Effect of macromolecular composition of micro-organisms on the thermodynamic description of their growth.
Biochim. Biophys. Acta
936:406-412[Medline].
|
| 17.
|
Ng, L. T.,
A. Pascaud, and M. Pascaud.
1987.
Hydrochloric acid hydrolysis of proteins and determination of tryptophan by reversed-phase high-performance liquid chromatography.
Anal. Biochem.
167:47-52[CrossRef][Medline].
|
| 18.
|
Novák, L.,
M. Cocaign-Bousquet,
N. D. Lindley, and P. Loubière.
1997.
Metabolism and energetics of Lactococcus lactis during growth in complex or synthetic media.
Appl. Environ. Microbiol.
63:2665-2670[Abstract].
|
| 19.
|
Otto, R.,
B. Ten Brink,
H. Veldkamp, and W. N. Konings.
1983.
The relation between growth rate and electrochemical proton gradient of Streptococcus cremoris.
FEMS Microbiol. Lett.
16:69-74.
|
| 20.
|
Paalme, T.,
A. Olivson, and R. Vilu.
1982.
13C-NMR of CO2-fixation during the heterotrophic growth in Chlorobium thiosulfatophilum.
Biochim. Biophys. Acta
720:311-319[CrossRef].
|
| 21.
|
Peterson, G.
1983.
Determination of total proteins.
Methods Enzymol.
91:95-119[Medline].
|
| 22.
|
Plihon, F.,
P. Taillandier, and P. Strehaiano.
1996.
Oxygen effect on lactose catabolism by a Leuconostoc mesenteroides strain: modeling of general O2-dependent stoichiometry.
Biotech. Bioeng.
49:63-69.
|
| 23.
|
Poolman, B., and W. N. Konings.
1988.
Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport.
J. Bacteriol.
170:700-707[Abstract/Free Full Text].
|
| 24.
|
Poolman, B.,
E. Smid,
H. Veldkamp, and W. N. Konings.
1987.
Bioenergetic consequences of lactose starvation for continuously cultured Streptococcus cremoris.
J. Bacteriol.
169:1460-1468[Abstract/Free Full Text].
|
| 25.
|
Saurina, J., and S. Hernandez-Cassou.
1994.
Determination of amino acids by ion-pair liquid chromatography with post-column derivatization using 1,2-naphthoquinone-4-sulfonate.
J. Chromatogr.
676:311-319[CrossRef].
|
| 26.
|
Schleifer, K. H.,
J. Kraus,
C. Dvorak,
R. Kilpper-Bälz,
M. D. Collins, and W. Fischer.
1985.
Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen. nov.
Syst. Appl. Microbiol.
6:183-195.
|
| 27.
|
Schmitt, P.,
C. Diviès, and R. Cardona.
1992.
Origin of end-products from the co-metabolism of glucose and citrate by Leuconostoc mesenteroides subsp. cremoris.
Appl. Microbiol. Biotechnol.
36:679-683.
|
| 28.
|
Schulze, U.,
G. Liden,
J. Nielsen, and J. Villadsen.
1996.
Physiological effects of nitrogen starvation in an anaerobic batch culture of Saccharomyces cerevisiae.
Microbiology
142:2299-2310[Abstract/Free Full Text].
|
| 29.
|
Sivakanesan, R., and E. A. Dawes.
1980.
Anaerobic glucose and serine metabolism in Staphylococcus epidermidis.
J. Gen. Microbiol.
118:143-157[Abstract/Free Full Text].
|
| 30.
|
Terzaghi, B., and W. E. Sandine.
1975.
Improved medium for lactic streptococci and their bacteriophages.
Appl. Microbiol.
29:807-813.
|
| 31.
|
Thomas, T. D., and R. D. Batt.
1969.
Degradation of cell constituents by starved Streptococcus lactis in relation to survival.
J. Gen. Microbiol.
58:347-362[Abstract/Free Full Text].
|
| 32.
|
Vallino, J., and G. Stephanopoulos.
1993.
Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction.
Biotech. Bioeng.
41:633-646[CrossRef].
|
| 33.
|
Wann, S. R.,
P. T. Thorsen, and M. Kreevoy.
1981.
Reduction of carboxylic acids derivatives by BH4 in acidic dimethyl sulfoxide.
J. Org. Chem.
46:2579-2581[CrossRef].
|
| 34.
|
Wanner, U., and T. Egli.
1990.
Dynamics of microbial growth and cell composition in batch culture.
FEMS Microbiol. Rev.
75:19-44[CrossRef].
|
| 35.
|
Yano, H.,
K. Aso, and A. Tsugita.
1990.
Further study on gas phase acid hydrolysis of protein: improvement of recoveries for tryptophan, tyrosine and methionine.
J. Biochem.
108:579-582[Abstract/Free Full Text].
|
| 36.
|
Yvon, M.,
S. Thirouin,
L. Rijnen,
D. Fromentier, and J. C. Gripon.
1997.
An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.
Appl. Environ. Microbiol.
63:414-419[Abstract].
|
Journal of Bacteriology, February 2000, p. 1136-1143, Vol. 182, No. 4
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Teusink, B., Wiersma, A., Molenaar, D., Francke, C., de Vos, W. M., Siezen, R. J., Smid, E. J.
(2006). Analysis of Growth of Lactobacillus plantarum WCFS1 on a Complex Medium Using a Genome-scale Metabolic Model. J. Biol. Chem.
281: 40041-40048
[Abstract]
[Full Text]
-
Raynaud, S., Perrin, R., Cocaign-Bousquet, M., Loubiere, P.
(2005). Metabolic and Transcriptomic Adaptation of Lactococcus lactis subsp. lactis Biovar diacetylactis in Response to Autoacidification and Temperature Downshift in Skim Milk. Appl. Environ. Microbiol.
71: 8016-8023
[Abstract]
[Full Text]
-
Goffman, F. D., Alonso, A. P., Schwender, J., Shachar-Hill, Y., Ohlrogge, J. B.
(2005). Light Enables a Very High Efficiency of Carbon Storage in Developing Embryos of Rapeseed. Plant Physiol.
138: 2269-2279
[Abstract]
[Full Text]
-
Netzer, R., Peters-Wendisch, P., Eggeling, L., Sahm, H.
(2004). Cometabolism of a Nongrowth Substrate: L-Serine Utilization by Corynebacterium glutamicum. Appl. Environ. Microbiol.
70: 7148-7155
[Abstract]
[Full Text]
-
Palmfeldt, J., Paese, M., Hahn-Hagerdal, B., van Niel, E. W. J.
(2004). The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis. Appl. Environ. Microbiol.
70: 5477-5484
[Abstract]
[Full Text]
-
Vido, K., le Bars, D., Mistou, M.-Y., Anglade, P., Gruss, A., Gaudu, P.
(2004). Proteome Analyses of Heme-Dependent Respiration in Lactococcus lactis: Involvement of the Proteolytic System. J. Bacteriol.
186: 1648-1657
[Abstract]
[Full Text]
-
Nordkvist, M., Jensen, N. B. S., Villadsen, J.
(2003). Glucose Metabolism in Lactococcus lactis MG1363 under Different Aeration Conditions: Requirement of Acetate To Sustain Growth under Microaerobic Conditions. Appl. Environ. Microbiol.
69: 3462-3468
[Abstract]
[Full Text]
-
Neves, A. R., Ventura, R., Mansour, N., Shearman, C., Gasson, M. J., Maycock, C., Ramos, A., Santos, H.
(2002). Is the Glycolytic Flux in Lactococcus lactis Primarily Controlled by the Redox Charge? KINETICS OF NAD+ AND NADH POOLS DETERMINED IN VIVO BY 13C NMR. J. Biol. Chem.
277: 28088-28098
[Abstract]
[Full Text]
-
Andersen, H. W., Solem, C., Hammer, K., Jensen, P. R.
(2001). Twofold Reduction of Phosphofructokinase Activity in Lactococcus lactis Results in Strong Decreases in Growth Rate and in Glycolytic Flux. J. Bacteriol.
183: 3458-3467
[Abstract]
[Full Text]
Top
Abstract
Introduction
