Thông tin tài liệu
Expression of the
pyrG
gene determines the pool sizes of CTP
and dCTP in
Lactococcus lactis
Casper M. Jørgensen, Karin Hammer, Peter R. Jensen and Jan Martinussen
Bacterial Physiology and Genetics, BioCentrum-DTU, Technical University of Denmark, Kgs. Lyngby, Denmark
The pyrG gene from Lactococcus lactis encodes CTP syn-
thase (EC 6.4.3.2), an enzyme converting UTP to CTP. A
series of strains were constructed with different levels of
pyrG expression by insertion of synthetic constitutive pro-
moters with different strengths in front of pyrG.These
strains expressed pyrG levels in a range from 3 to 665%
relative to the wild-type expression level. Decreasing the level
of CTP synthase to 43% had no effect on the growth rate,
showing that the capacity of CTP synthase in the cell is
in excess in a wild-type strain. We then studied how pyrG
expression affected the intracellular pool sizes of nucleotides
and the correlation between pyrG expression and nucleotide
pool sizes was quantified using metabolic control analysis
in terms of inherent control coefficients. At the wild-type
expression level, CTP synthase had full control of the CTP
concentration with a concentration control coefficient close
to one and a negative concentration control coefficient of
)0.28 for the UTP concentration. Additionally, a concen-
tration control coefficient of 0.49 was calculated for the
dCTP concentration. Implications for the homeostasis of
nucleotide pools are discussed.
Keywords: pyrG; CTP synthase; metabolic control analysis;
metabolism; EC 6.4.3.2.
1
Synthesis of ribonucleotides and deoxyribonucleotides is
an essential part of cellular metabolism, as synthesis of
RNA requires ribonucleotides and DNA replication is
dependent on deoxyribonucleotides. The involvement of
nucleotides in these central cellular pathways suggests that
it is important for the cell to control the synthesis of
nucleotides and to be able to maintain a steady supply of
these essential precursors either by de novo biosynthesis or
by uptake of precursors from the growth medium. In
addition, the involvement of nucleotides in regulatory
processes such as regulation of gene expression and
modulation of kinetic properties of enzymes emphasizes
the need for tight regulation of the level of nucleotides in
the cell. Indeed, expression of genes responsible for the
de novo biosynthesis of ribonucleotides in Gram-positive
bacteria such as Lactococcus lactis and Bacillus subtilis are
regulated by the availability of purines and pyrimidines.
The pyrimidine biosynthetic genes are regulated by the
RNA-binding regulatory protein PyrR that regulates gene
expression by an attenuation mechanism through sensing
of the UMP concentration in the cell [1–5]. However,
PyrR is not involved in the regulation of expression of the
pyrG gene encoding CTP synthase (EC 6.4.3.2) in L. lactis
and B. subtilis,aspyrG expression is probably regulated
by an attenuation mechanism responding to the CTP
concentration in the cell [6,7]. The reaction catalyzed by
CTPsynthase(UTP+glutamine+ATP fi CTP +
glutamate + ADP + P
i
) involves all four ribonucleo-
tides; UTP and CTP are substrate and product, respect-
ively, ATP is used as an energy source and GTP is an
allosteric activator of the reaction [8]. CTP synthase has
a central role in pyrimidine metabolism, as the enzyme
catalyses the only reaction resulting in the amination of
the pyrimidine ring into a cytosine derivative (Fig. 1). It is
therefore of interest to examine to what extent this
enzyme controls the fluxes and metabolite concentrations
in the pathway. Here, we have determined the importance
of CTP synthase for growth rate, the concentration of
ribonucleotides and for the concentration of the deoxy-
ribonucleotide dCTP using the methods developed for
metabolic control analysis [9,10]. We show that CTP
synthase has a strong inherent control on the CTP and
dCTP concentrations and a negative control on the UTP
concentration.
Materials and methods
Bacterial strains and plasmids
The strains and plasmids used in this study are listed in
Table 1. Plasmid pCJ31B contains the L. lactis pyrG gene,
and was made from a PCR-product made with prim-
ers pyrG11a (5¢-GTAGAAGCTAAAATCTGG-3¢)and
SLLH7 (5¢-TACAAAAGATTTTGGGC-3¢) cloned in
the TOPO TA cloning kit from Invitrogen. Chromosomal
DNA purified from MG1363 was used as a template for the
PCR amplification.
Growth medium and growth conditions
L. lactis strains were grown either in M17 broth supplied
with 1% (w/v) glucose or in defined SA medium [11] with
Correspondence to J. Martinussen, BioCentrum-DTU, Bacterial
Physiology and Genetics, Technical University of Denmark, Building
301, DK-2800 Kgs. Lyngby, Denmark. Fax: + 45 45932809,
Tel.: + 45 45252498, E-mail: jma@biocentrum.dtu.dk
Enzyme: CTP synthase (EC 6.4.3.2).
(Received 9 January 2004, revised 14 April 2004,
accepted 16 April 2004)
Eur. J. Biochem. 271, 2438–2445 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04168.x
1% (w/v) glucose at 30 °C in 50-mL plastic tubes without
aeration. Erythromycin was added to a concentration of
2 lgÆmL
)1
, chloramphenicol to 5 lgÆmL
)1
. When needed,
cytidine was added to 20 lgÆmL
)1
in SA medium and to
1000 lgÆmL
)1
in M17 medium.
Transformation of DNA to
L. lactis
L. lactis cells were transformed by electroporation as
described by [12]. Following transformation, the cells were
incubated for 2 h in M17 medium with glucose and cytidine
at 1000 lgÆmL
)1
for phenotypic expression.
Isolation of chromosomal DNA from
L. lactis
Chromosomal DNA was isolated as described in [13].
Isolation of strains with altered
pyrG
expression
A PCR product was made with the primers pyrGCP2
(5¢-ACGCTCGAGATNNNNNAGTTTATTCTTGACA
NNNNNNNNNNNNNNNNNTATAATNNNNCCTC
TGGGGAGCTGTTTTTG-3¢) and pyrG13b (5¢-GCTGA
ACTGCAGAACTCCTGAGTTAAGGAGAG-3¢)using
pCJ31B as template and the Elongase enzyme mix (Life
Technologies). The pyrGCP2 primer was designed in such a
way that the 3¢ end would anneal to the template upstream
of the pyrG open reading frame but after the terminator in
the attenuator. The downstream primer is located before the
pyrG terminator, but after the stop codon. The PCR
product was digested with XhoIandPstI and ligated in
plasmidpLB86alsodigestedwithXhoIandPstI. After
ligation, the plasmids were transformed to CJ327 (a
pyrG::ISS1, cdd strain with plasmid pLB65) and plated on
Table 1. Strains and plasmids used in this study.
Strain or plasmid Relevant description
Reference
or source
L. lactis cells
MG1363 L. lactis ssp. cremoris strain [33]
MB109 MG1363 cdd [34]
CJ233 MB109/pAK80 [6]
CJ295 MB109 pyrG::ISS1 [6]
CJ327 CJ295/pLB65 This study
CJ340 CJ327 with pLB86 integrated in attB site This study
CJ381 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ382 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ383 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ388 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ405 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ406 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ407 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ410 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ411 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ413 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ418 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
CJ420 CJ327 with pyrG gene transcribed from synthetic promoter integrated in attB site This study
Plasmids
pCJ31B Contains pyrG gene and promoter in pCR2.1-TOPO vector This study
pLB65 Expresses the bacteriophage TP901-1 integrase protein; carries chloramphenicol resistance gene [17]
pLB86 Integration vector with promoter-less reporter genes lacLM, erythromycin resistance gene and attP site [17]
pCR2.1-TOPO Plasmid vector Invitrogen
pAK80 Contains promoterless lacLM genes [14]
Fig. 1. Simplified representation of pyrimidine nucleotide metabolism in
L. lactis. Only reactions relevant for this study are included in the
figure. The central part of the figure shows the conversion of UTP to
CTP catalyzed by CTP synthase encoded by pyrG. Involvement of
pyrimidine nucleotides in synthesis of DNA, RNA, and phospholipids
are indicated. Breakdown of mRNA is indicated by broken arrows.
For more details on nucleotide metabolism in Gram-positive bacteria,
see previously published review [32]. Gene symbols refer to the fol-
lowing proteins: cdd, cytidine deaminase; nrdEF, aerobic ribonucleo-
tide reductase; nrdDG, anaerobic ribonucleotide reductase.
Ó FEBS 2004 pyrG controls [CTP] and [dCTP] in L. lactis (Eur. J. Biochem. 271) 2439
M17 plates with glucose, erythromycin, chloramphenicol,
cytidine at 1000 lgÆmL
)1
and 5-bromo-4-chloro-3-indolyl-
b-
D
-galactoside (X-gal) at 90 lgÆmL
)1
.
Growth experiments
The strains were grown over night at 30 °ConSAplates
containing glucose, erythromycin and cytidine. Several
colonies from the plates were inoculated in 1 mL SA
medium with glucose, erythromycin and cytidine and grown
for 5–6 h at 30 °C. This growing culture was diluted and
used to inoculate 10 mL of SA medium with glucose,
erythromycin and cytidine so that the next day the growth
experiment could start with an exponentially growing
culture (D
436
<0.8)
2
. In order to remove the cytidine
present in the overnight culture, the cells were centrifuged
for 15 min at 3000 g
3
, washed twice with 0.9% (w/v) NaCl
and resuspended in 2 mL of 0.9% (w/v) NaCl. Fifty
milliliters of SA medium with glucose and erythromycin was
inoculated to D
436
of 0.025 and the growth monitored by
measuring D
436
.AtD
436
of 0.8, 35 mL of the culture was
harvested, washed once with 0.9% (w/v) NaCl and resus-
pended in 1 mL of Z buffer.
b-Galactosidase measurement
b-Galactosidase activity in exponentially grown cells was
determined at 30 °C as previously described [14], except
that cell density was measured at 436 nm and the specific
activity was therefore determined as A
420
/D
436
per minute
per mL of culture (units/D
436
).
Nucleotide pool determinations
The concentration of nucleotides in the cell was determined
by thin layer chromatography on PEI-plates of
33
PO
43
-
labeled nucleotides extracted from exponentially growing
cells at a cell density of D
436
¼ 0.8 as described previously
[15].
Determination of concentration control coefficients
The experimental data of reporter gene activity from the
strains with altered pyrG expression and corresponding
growth rate or concentrations of (deoxy)ribonucleo-
tides were plotted and fitted to functions using the
software program
GRAFIF
(Erithacus Software Ltd,
Harley, Surrey, UK). Wild-type b-galactosidase expres-
sion level was obtained by plotting the relative CTP,
UTP, and dCTP concentrations against the specific
b-galactosidase activity (z) in units/D
436
4
and fitting to
the functions f(z) ¼ 2.49–2.49 · exp() 0.0237 · z
1.362
)for
CTP, f(z) ¼ 1.55–0.268 · ln(z) for UTP, and f(z) ¼ 1.27–
1.27 · exp()0.131 · z)
5
for dCTP. The average of the
b-galactosidase activities obtained for the three nucleo-
tides at the wild-type pool level was taken as wild-type
b-galactosidase expression level of 9.5 units/D
436
.The
CTP concentration data were then fitted to the func-
tion f(x) ¼ 3.81–3.81 · exp()0.00101 · x
1.352
), where x is
expression of the reporter genes relative to the wild-type
level of 9.5 units/D
436
. When the CTP concentration data
were fitted to the function f(x) ¼ 0.0068 · 0.998x · x
1.225
,
a similar concentration control coefficient was obtained
at the wild-type level. To determine control of pyrG
expression on the UTP concentration, the data were
fitted to two functions: f(x) ¼ 9.20–1.132 · ln(x) and
f(x) ¼ 10.06 · 0.999x · x-0.191. Both functions gave sim-
ilar concentration control coefficients at the wild-type
level. The ATP and GTP concentrations were fitted to
the linear functions f(x) ¼ 7.81–0.00342 · xandf(x)¼
1.98–0.00176 · x. The change in dCTP concentration
was found to fit the equation f(x) ¼ 0.614 · [1 –
exp()0.0128 · x)]; fitting the data to a quadratic equa-
tion [f(x) ¼ 0.198 + 0.00236 · x ) 0.00000266 · x2) did
not affect the calculated concentration control coefficient
at the wild-type level. Control coefficients for CTP
synthase (PyrG) on the concentration of (deoxy)ribonu-
cleotides ([NTP]) were calculated from the equation ¼
fd([NTP])/[NTP]g/fd(b-gal)/b-galg. The growth rate
data were fitted to the function f(x) ¼ 0.650 · [1 –
exp() 0.2197 · x)], although fitting the data to the
function f(x) ¼ 0.664 · 0.1061/x gave almost identical
control coefficients except at very low x values.
Results
Isolation of strains with altered
pyrG
expression
Strains with different constitutive expression levels of the
6
pyrG gene encoding CTP synthase were isolated using a
PCR strategy where the pyrG gene under control of
synthetic constitutive promoters was integrated on the
chromosome of L. lactis [16]. A degenerate primer con-
taining a promoter with consensus )10 and )35 promoter
regions separated by a randomized spacer of 17 nucleo-
tidesplusa3¢-end with homology to the upstream
sequence from pyrG and a primer with homology to a
region downstream of pyrG were used to generate PCR
products covering the entire pyrG gene downstream of
synthetic promoters. Expression from pyrG in the wild
type is regulated by the concentration of CTP in the cell
by an attenuation mechanism in the 5¢-end of the pyrG
mRNA [6]. The primer was constructed so the PCR
product did not contain these regulatory signals to obtain
constitutive expression. The downstream primer was
designed in such a way that, after the pyrG open-reading
frame, the terminator could not be located on the PCR
product. The obtained DNA fragment was inserted in the
integration vector pLB86, which contains an erythromycin
resistance marker, the lacLM reporter genes as well as the
attachment site attP from the bacteriophage TP901-1. In
the presence of the TP901-1 integrase, pLB86 will insert
with high frequency at the attB site on the chromosome;
the promoter library was therefore transformed to a strain
carrying plasmid pLB65 expressing the integrase [17]. The
promoter fusions were integrated on the chromosome of
the CTP synthase deficient strain CJ295 (pyrG::ISS1),
which also carries a mutation in the cdd gene encoding
cytidine deaminase (Fig. 1) in order to prevent degrada-
tion of cytidine added to the growth medium. The
transformants were selected on rich media with a cytidine
concentration of 1000 lgÆmL
)1
even though we have
previously isolated pyrG mutants on defined media with
only20or50lg of cytidine per milliliter [6,8]. However,
2440 C. M. Jørgensen et al. (Eur. J. Biochem. 271) Ó FEBS 2004
L. lactis pyrG mutants do not grow on M17 media with
cytidine at such low concentrations. Apparently, com-
pounds present in M17 medium inhibits cytidine metabo-
lism, most likely by inhibiting the uptake of cytidine [15].
Adding cytidine in excess at 1000 lgÆmL
)1
relieves the
inhibitory effect and allows for growth of L. lactis pyrG
mutants on rich medium. More than 1500 colonies
with potentially different pyrG expression levels were
isolated. Forty strains with colors of colonies ranging
from white to blue on X-gal indicator plates were purified,
and after an initial screening of b-galactosidase activity,
12 strains were selected for further analysis. The pyrG
gene from these strains was amplified by PCR and
sequenced in order to exclude strains having mutations
in the pyrG gene.
Estimation of
pyrG
expression from b-galactosidase
activity
The 12 selected strains were grown in defined medium
without cytidine and harvested during exponential growth
for determination of the level of pyrG expression. Several
attempts to measure CTP synthase activity in L. lactis
crude extracts using an assay based on the conversion of
UTP to N
4
-hydroxy-CTP in the presence of hydroxyl-
amine [18] was unfortunately unsuccessful. This is in
accordance with earlier results from B. subtilis, where no
CTP synthase activity could be detected in crude extracts
[7]. The estimation of pyrG expression in the constructed
strains was therefore based on reporter gene activity, as
b-galactosidase is expressed in an operon with pyrG.The
specific b-galactosidase activity in the selected group of
strains with altered pyrG expression was determined and
varied over a 20-fold range from 0.3 to 63 units/D
436
.The
nucleotide pool sizes in the 12 strains were determined,
and it was found that the CTP, UTP and dCTP pools
were affected by the pyrG expression level. Figure 2 shows
the correlation between the nucleotide concentrations and
the specific b-galactosidase activity in the range from 4 to
25 attenuance units. The nucleotide pool sizes are shown
relative to the wild-type concentrations from an isogenic
strain carrying a wild-type pyrG gene (CJ233). From the
wild-type concentrations of the three nucleotides in Fig. 2,
it can therefore be estimated that a specific activity of
b-galactosidase of 9.5 units/attenuance results in the same
nucleotide concentrations found in a wild-type strain. A
specific b-galactosidase activity of 9.5 units/D
436
was
therefore taken as the reference level where the CTP
synthase activity is the same as in a wild-type cell.
CTP synthase has no control on the growth rate
of
L. lactis
The strains with altered expression of pyrG were grown in
defined medium without cytidine and the specific growth
rate was determined. The b-galactosidase activity in each
strain was calculated relative to 9.5 units/D
436
,which
reflects the wild-type CTP synthase level. The strains then
have b-galactosidase activities ranging from 3% of the
wild-type level to 6.6-fold increased expression. Figure 3
shows the specific growth rate of the strains with altered
expression of pyrG plotted against the relative b-galac-
tosidase activity. No change in growth rate was observed
around the wild-type level and only when the b-galactosi-
dase activity dropped below 40% of the wild-type level,
the growth rate was affected. From the curve fit in Fig. 3,
control coefficients for pyrG expression on the specific
growth rate for all lacLM levels were determined. The
level of CTP synthase had zero control on the growth rate
in the range from 40 to 600% relative b-galactosidase
expression; the control increased to more than 0.5 at
expression levels below 5% of the wild-type level, reflect-
ing that CTP synthase is an essential enzyme for growth
of L. lactis.
(Deoxy)ribonucleotide pool sizes in mutants with
different levels of
pyrG
expression
Figure 4A shows the variation of the CTP concentration as
a function of pyrG expression measured as b-galactosidase
activity from the lacLM reporter genes. There is a clear
correlation between expression of lacLM and the CTP pool
Fig. 2. Correlation of specific b-galactosidase activity and concentra-
tions of the nucleotides CTP, dCTP, and UTP. Concentrations of CTP,
UTP and dCTP relative to the wild type are plotted against the specific
b-galactosidase activity in strains with modulated pyrG expression.
The horizontal stippled line indicates the wild-type nucleotide con-
centration, which has been set to one for all three nucleotides.
Fig. 3. Specific growth rate and control coefficient for CTP synthase.
The specific growth rate is shown as function of b-galactosidase
activity from mutants with altered pyrG expression given as percent
relative to the wild-type activity. The curve fitted to the experimental
data is shown as a thin line; the thick black line indicates the calculated
control coefficient.
Ó FEBS 2004 pyrG controls [CTP] and [dCTP] in L. lactis (Eur. J. Biochem. 271) 2441
size. Increased reporter gene expression to 660% of the wild-
type level results in an increase in the CTP concentration of
almost 2.5-fold; decreased expression results in a sevenfold
decrease in the CTP pool size. With respect to the UTP pool
size, the correlation appeared to be inverse (Fig. 4B), with
UTP decreased more than twofold at increased CTP
synthase activity and increased twofold at low activity. No
significant correlation was observed for the purine nucleo-
tides ATP and GTP, although there is a tendency for the
concentration of these nucleotides to decrease with increas-
ing pyrG expression (Fig. 4C,D). The variation of the
concentration of the deoxyribonucleotide dCTP with
respect to altered pyrG expression is shown in Fig. 5. The
pattern of changes in dCTP pool size resembles the one for
CTP. No significant changes in the concentrations of the
deoxyribonucleotides dATP, dGTP and dTTP were
observed with different levels of pyrG expression (data not
shown).
CTP synthase has a positive control on the CTP and dCTP
concentrations and negative control on the UTP
concentration
The primary data on Figs 4 and 5 already indicates that the
level of CTP synthase is important for the pool sizes of CTP,
UTP and dCTP. In order to calculate how important the
Fig. 5. Effects of changing pyrG expression on the concentration of
dCTP. The concentration of dCTP is shown as a function of relative
b-galactosidase activity in percent of the wild-type level. The concen-
tration of the deoxyribonucleotide is given in nanomoles per mg (dry
weight). The experimental data are fitted to the thin black line; the
calculated concentration control coefficient is shown as a thick black
line. The dCTP pool size in the reference strain CJ233 is shown by a
stippled line.
Fig. 4. Effects of changing pyr G expression on the concentrations of
ribonucleotides. The concentrations of CTP, UTP, GTP and ATP (A,
B, C, and D) are shown as functions of relative b-galactosidase activity
in percent of the wild-type level. The concentrations of the ribo-
nucleotides are given in nmolÆmg
)1
(dry weight). The experimental
data are fitted to the thin black lines; the calculated concentration
control coefficients for CTP and UTP are shown as thick black lines.
Ribonucleotide pool sizes in the reference strain CJ233 are shown by
horizontal stippled lines.
2442 C. M. Jørgensen et al. (Eur. J. Biochem. 271) Ó FEBS 2004
enzyme activity is, we used metabolic control analysis to
quantify the effect of a sustained modulation of CTP
synthase, i.e. in terms of so-called inherent control coeffi-
cients [19,20]. The curve fits shown in Fig. 4A,B were used
for calculation of the concentration control coefficients for
CTP synthase on the concentrations of CTP and UTP in
L. lactis. CTP synthase (PyrG) has a high inherent control
on the CTP concentration, as at the wild-type level the
calculated concentration control coefficient C
½CTP
PyrG
is 1.03
(Fig. 4A). The control coefficient increased to 1.35 at very
low expression of the pyrG gene and decreased to zero at
high expression levels. The concentration control coefficient
on the UTP concentration was calculated from two different
curve fits, with almost identical results. The control
coefficients in Fig. 4B is from the equation [UTP](x) ¼
9.20–1.13 · ln(x). The concentration control coefficient
decreased from )0.11 at very low expression of pyrG to
)0.28 at the wild-type level and to )0.63 at high expression
levels. CTP synthase was found to have control on not only
the CTP concentration but also the dCTP concentration.
At the wild-type level, the concentration control coefficient
was calculated to ¼ 0.49 (Fig. 5). At low expression levels,
this value increased to one and decreased to zero at high
expression levels.
Strains with decreased CTP and dCTP concentrations
have reduced growth at 15 °C
In Escherichia coli, deletion of the cmk gene encoding
cytidine monophosphate kinase, results in reduced CTP
and dCTP pool sizes, probably due to impaired reutiliza-
tion of nucleotides generated from, e.g. mRNA turnover
[21], and an interesting phenotype of the E. coli cmk
mutant is an inability to grow at low temperatures. As
some of the isolated L. lactis strains with altered pyrG
expression have very low CTP and dCTP pool sizes, they
were tested for growth on solid medium at 15 °Cand
30 °C. Table 2 compares the growth of seven strains with
reduced pyrG expression to the growth of a pyrG::ISS1
mutant with a vector inserted in the attB site (CJ340) as
well as to the growth of a pyrG + strain (CJ233). The
three strains with the lowest pyrG expression level, and
thus the lowest CTP and dCTP concentrations in the cell,
grew significantly slower at 15 °Cthanat30°C compared
to strains with normal pyrG expression. One of these
strains has 43% pyrG expression relative to the wild-type
andnogrowthdefectat30°C. However, the two strains
with the lowest pyrG expression levels at 3–4% of the
wild-type level (CJ381 and CJ388) also showed reduced
growth rate at 30 °C compared to the wild-type, but these
strains are growth impaired at 15 °C, as they do not grow,
even after 8 days of incubation at 15 °C. Addition of
cytidine to the growth medium restores growth, suggesting
that the reduced growth rate is indeed related to the
decreased CTP and dCTP pool sizes.
Discussion
In this work, we have modulated the expression of the
L. lactis pyrG gene encoding CTP synthase. To our
knowledge, this is the first time an enzyme in nucleotide
metabolism has been the subject of metabolic control
analysis. We have established that the level of CTP synthase
has no control on the growth rate at the wild-type level in
L. lactis. At highly reduced pyrG expression, CTP synthase
controls the growth rate, thus confirming that CTP synthase
is an essential enzyme for growth of L. lactis in the absence
of cytidine.
CTP synthase has a high positive control on the
concentrations of CTP and dCTP and a negative control
on the UTP concentration. dCTP is synthesized from CTP
by a reaction catalyzed by ribonucleotide reductase, which
converts the ribose part to 2¢-deoxyribose. In L. lactis,two
ribonucleotide reductases have been identified: NrdDG,
required for strict anaerobic growth and NrdEF that does
not function in the absence of oxygen [22,23] (Fig. 1). The
two lactococcal ribonucleotide reductases have different
substrate specificity as NrdDG is an NTP reductase and
NrdEF is an NDP reductase. Although the data in Figs 4A
and 5 show that the dCTP concentration varies in a similar
way as the CTP concentration when pyrG expression is
altered, it is not possible from the available data to conclude
whether reduction occurs at the tri- or diphosphate level
in our experiments.
Table 2. Growth of strains with modulated pyrG expression at 15 °C and 30 °C. The relative pyrG expression is given as per cent of wild-type
b-galactosidase activity. The activity in the pyrG strain CJ340 is defined as 0%; activity in the pyrG
+
strain CJ233 is defined as 100%. Growth was
on solid defined medium with erythromycin at 2 lgÆmL
)1
and 1% glucose as carbon source in the presence or absence of cytidine (20 lgÆmL
)1
). 3/4,
no growth; +++, good growth after 4 days at 15 °C or two days at 30 °C.
Strain Genotype
Relative pyrG
expression (%)
Growth at 15 °C Growth at 30 °C
None Cytidine None Cytidine
CJ406 cdd pyrG::ISS1 attB::pyrG
+
99 +++ +++ +++ +++
CJ418 cdd pyrG::ISS1 attB::pyrG
+
89 +++ +++ +++ +++
CJ410 cdd pyrG::ISS1 attB::pyrG
+
88 +++ +++ +++ +++
CJ405 cdd pyrG::ISS1 attB::pyrG
+
77 +++ +++ +++ +++
CJ413 cdd pyrG::ISS1 attB::pyrG
+
43 + +++ +++ +++
CJ381 cdd pyrG::ISS1 attB::pyrG
+
4.3 3/4 +++ + +++
CJ388 cdd pyrG::ISS1 attB::pyrG
+
3.3 3/4 +++ + +++
CJ340 cdd pyrG::ISS1 attB::pLB86 0 3/4 +++ 3/4 +++
CJ233 cdd 100 +++ +++ +++ +++
Ó FEBS 2004 pyrG controls [CTP] and [dCTP] in L. lactis (Eur. J. Biochem. 271) 2443
The finding that CTP synthase has no control on the
growth rate but a strong control on the CTP and dCTP
concentrations is perfectly in line with the theory of
metabolic supply and demand analysis [24]. This theory
predicts that for biosynthetic pathways, such as those
leading to the biosynthesis of amino acids or nucleotides,
the control of the flux should reside in the demand for the
end-product, whereas the supply determines the degree of
concentration control.
Recently it was found that DNA supercoiling in E. coli
is under tight homeostatic control with 87% of imposed
changes being counteracted by homeostatic mechanisms
[19,20]. The homeostasis was found to take place at the
metabolic and genetic level with 72 and 28%, respectively.
Here it is important to remember that in the analysis of
CTP synthase, the feedback mechanism that may have
acted on the level of pyrG expression has been removed.
The strength of the feedback regulation of CTP on pyrG
expression, i.e. the elasticity of pyrG expression for the
CTP concentration [25], has not been quantified, but the
regulation appears to be quite strong: when a pyrG
mutant was starved for cytidine the CTP pool dropped
more than 10-fold and at the same time the expression of
a reporter gene fusion to the pyrG promoter increased
37-fold [6]. Therefore, the control exerted by CTP
synthase in a ÔnormalÕ cell, i.e. where the regulation of
pyrG expression is operative, could very well differ
significantly from the control measured in the current
study, where the elasticity of pyrG expression is zero. The
control coefficients we have obtained by modulating the
level of transcription are called inherent control coeffi-
cients [19,20]. In the experiments, product inhibition by
CTP on CTP synthase was intact, and this feedback
inhibition [8] was shown to be unable to fully counteract
an increase in the CTP concentration in strains with
increased pyrG expression of up to 250% of the wild-type
level (Fig. 4A). The results show that the feedback
inhibition of the CTP synthase enzyme is incomplete
in vivo. In conclusion, the homeostasis of the CTP pool in
the wild-type cell is primarily a matter of regulation of
pyrG expression exerted by the attenuator found immedi-
ately in front of the pyrG open reading frame.
Strains with pyrG expression from 43% to 665% of the
wild-type level have growth rates at the wild-type level at
30 °C, implying that the need for CTP in these strains is
similar to the wild-type. This suggests that for strains with
pyrG expression of 43%, the average flow of substrate
through each CTP synthase enzyme is increased approxi-
mately 2.5-fold and that the average in vivo activity of
CTP synthase is correspondingly increased. Increased
in vivo activity of CTP synthase may be due to the reduced
CTP concentration, as the L. lactis CTP synthase enzyme is
feedback inhibited by CTP [8], as well as due to an increase
in the substrate concentration.
It was not possible to detect CTP synthase activity in
L. lactis cell extracts, and determination of pyrG expression
in the constructed strains with altered pyrG expression was
therefore dependent on b-galactosidase activity measure-
ments. As a clear correlation was observed between
nucleotide pool sizes and b-galactosidase activity, the
wild-type b-galactosidase activity was established using the
in vivo concentrations of nucleotides (Fig. 2). This deter-
mination is important, as calculations of control coefficients
are dependent on knowledge of enzyme activities. However,
even changing the estimate of the wild-type b-galactosidase
activity with 25% does not result in significant changes in
the concentration control coefficients. A variation of
± 25% in the wild-type b-galactosidase activity results in
changes in the concentration control coefficients for CTP
and UTP with less than 8% and less than 18% for dCTP
(data not shown).
A strain with pyrG expression reduced to 43% of the
wild-type level has decreased CTP and dCTP pool sizes and
show reduced growth at 15 °C, whereas no effect on growth
was observed at 30 °C (Table 2). The growth defect is
relieved by cytidine addition, suggesting that the observed
slow growth at 15 °C is a result of reduced CTP and dCTP
pool sizes. Bacteria grown at low temperatures have several
metabolic problems compared to growth at the optimal
growth temperature, including reduced enzyme activities,
low membrane fluidity, and decreased initiation of transla-
tion [26,27]. These factors may all be related to the impaired
growth at 15 °C of strains with decreased CTP and dCTP
pool sizes at 30 °C. Reduced synthesis or activity of CTP
synthase at 15 °C may affect the growth of cells with low
expression of pyrG, whereas cells with expression levels close
to the wild-type have excess CTP synthase capacity and are
thus not affected by a decrease in CTP synthase activity.
To maintain membrane structure and function, B. subtilis
change the fatty acid composition in the membrane during
cold shock [28,29]. Altered CTP and dCTP pool sizes may
result in perturbations of membrane synthesis, as both
nucleotides are used in the biosynthesis of phospholipids
through the synthesis of CDP-diacylglycerol from phos-
phatidic acid [30]. Dramatically changed CTP and dCTP
pool sizes in L. lactis may therefore inhibit the adaptation of
thecellmembranetogrowthat15°C resulting in slow
growth at this temperature. The importance of CTP
synthase in E. coli phospholipid biosynthesis was investi-
gatedinapyrG mutant starved for CTP by resuspending
cells in medium lacking cytidine thereby reducing the CTP
pool size. This results in accumulation of phosphatidic acid,
the substrate for CDP-diacylglycerol synthetase [31] and
increased resistance to the antibiotic erythromycin. How-
ever, the mechanisms for CTP and dCTP involvement in
lipid biosynthesis and adaptation to growth at low temper-
atures remain unclear.
Acknowledgements
We thank Martin Willemoe
¨
s for many helpful discussions. The
FØTEK Program and the DFFE supported this work through the
Centre for Advanced Food Studies.
References
1. Turner, R.J., Lu, Y. & Switzer, R.L. (1994) Regulation of the
Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an
autogenous transcriptional attenuation mechanism. J. Bacteriol.
176, 3708–3722.
2. Bonner, E.R., D’Elia, J.N., Billips, B.K. & Switzer, R.L. (2001)
Molecular recognition of pyr mRNA by the Bacillus subtilis
attenuation regulatory protein PyrR. Nucleic Acids Res. 29,
4851–4865.
2444 C. M. Jørgensen et al. (Eur. J. Biochem. 271) Ó FEBS 2004
3. Andersen, P.S., Martinussen, J. & Hammer, K. (1996) Sequence
analysis and identification of the pyrKDbF operon from Lacto-
coccus lactis including a novel gene, pyrK, involved in pyrimidine
biosynthesis. J. Bacteriol. 178, 5005–5012.
4. Martinussen, J. & Hammer, K. (1998) The carB gene encoding the
large subunit of carbamoylphosphate synthetase from Lacto-
coccus lactis is transcribed monocistronically. J. Bacteriol. 180,
4380–4386.
5. Martinussen, J., Schallert, J., Andersen, B. & Hammer, K. (2001)
The pyrimidine operon pyrRPB-carA from Lactococcus lactis.
J. Bacteriol. 183, 2785–2794.
6. Jørgensen, C.M., Hammer, K. & Martinussen, J. (2003) CTP
limitation increases expression of CTP synthase in Lactococcus
lactis. J. Bacteriol. 185, 6562–6574.
7. Meng, Q. & Switzer, R.L. (2001) Regulation of transcription of
the Bacillus subtilis pyrG gene, encoding cytidine triphosphate
synthetase. J. Bacteriol. 183, 5513–5522.
8. Wadskov-Hansen, S.L., Willemoe
¨
s, M., Martinussen, J.,
Hammer, K., Neuhard, J. & Larsen, S. (2001) Cloning and
verification of the Lactococcus lactis pyrG gene and characteri-
zation of the gene product, CTP synthase. J. Biol. Chem. 276,
38002–38009.
9. Heinrich, R., Rapoport, S.M. & Rapoport, T.A. (1977) Metabolic
regulation and mathematical models. Prog. Biophys. Mol. Biol. 32,
1–82.
10. Kacser, H. & Burns, J.A. (1973) The control of flux. In Rate
Control of Biological Processes (Davies, D.D., ed.), pp. 65–104.
Cambridge University Press, London.
11.Jensen,P.R.&Hammer,K.(1993)Minimalrequirementsfor
exponential growth of Lactococcus lactis. Appl. Environ. Micro-
biol. 59, 4363–4366.
12. Holo, H. & Nes, I.F. (1989) High-frequency transformation, by
electroporation, of Lactococcus lactis subsp. cremoris grown with
glycine in osmotically stabilized media. Appl. Environ. Microbiol.
55, 3119–3123.
13. Johansen, E. & Kibenich, A. (1992) Characterization of Leuco-
nostoc isolates from commercial mixed strain mesophilic starter
cultures. J. Dairy Sci. 75, 1186–1191.
14. Israelsen, H., Madsen, S.M., Vrang, A., Hansen, E.B. & Johansen,
E. (1995) Cloning and partial characterization of regulated pro-
moters from Lactococcus lactis Tn917-lacZ. integrants with the
new promoter probe vector, pAK80. Appl. Environ. Microbiol. 61,
2540–2547.
15. Martinussen, J., Wadskov-Hansen, S.L. & Hammer, K. (2003)
Two nucleoside uptake systems in Lactococcus lactis: competition
between purine nucleosides and cytidine allows for modulation of
intracellular nucleotide pools. J. Bacteriol. 185, 1503–1508.
16. Solem, C. & Jensen, P.R. (2002) Modulation of gene expression
made easy. Appl. Environ. Microbiol. 68, 2397–2403.
17. Brøndsted, L. & Hammer, K. (1999) Use of the integration ele-
ments encoded by the temperate lactococcal bacteriophage TP901-
1 to obtain chromosomal single-copy transcriptional fusions in
Lactococcus lactis. Appl. Environ. Microbiol. 65, 752–758.
18. Willemoe
¨
s, M. & Larsen, S. (2003) Substrate inhibition of Lac-
tococcus lactis cytidine 5¢-triphosphate synthase by ammonium
chloride is enhanced by salt-dependent tetramer dissociation.
Arch. Biochem. Biophys. 413, 17–22.
19. Jensen, P.R., van der Weijden, C.C., Jensen, L.B., Westerhoff,
H.V. & Snoep, J.L. (1999) Extensive regulation compromises the
extent to which DNA gyrase controls DNA supercoiling and
growth rate of Escherichia coli. Eur. J. Biochem. 266, 865–877.
20. Snoep, J.L., van der Weijden, C.C., Andersen, H.W., Westerhoff,
H.V. & Jensen, P.R. (2002) DNA supercoiling in Escherichia coli is
under tight and subtle homeostatic control, involving gene-
expression and metabolic regulation of both topoisomerase I and
DNA gyrase. Eur. J. Biochem. 269, 1662–1669.
21. Fricke,J.,Neuhard,J.,Kelln,R.A.&Pedersen,S.(1995)Thecmk
gene encoding cytidine monophosphate kinase is located in the
rpsA operon and is required for normal replication rate in
Escherichia coli. J. Bacteriol. 177, 517–523.
22. Jordan, A., Pontis, E., Aslund, F., Hellman, U., Gibert, I. &
Reichard, P. (1996) The ribonucleotide reductase system of Lac-
tococcus lactis. Characterization of an NrdEF enzyme and a new
electron transport protein. J. Biol. Chem. 271, 8779–8785.
23. Torrents, E., Buist, G., Liu, A., Eliasson, R., Kok, J., Gibert, I.,
Graslund, A. & Reichard, P. (2000) The anaerobic (class III)
ribonucleotide reductase from Lactococcus lactis: catalytic prop-
erties and allosteric regulation of the pure enzyme system. J. Biol.
Chem. 275, 2463–2471.
24. Hofmeyr, J.S. & Cornish-Bowden, A. (2000) Regulating the cel-
lular economy of supply and demand. FEBS Lett. 476, 47–51.
25. Burns, J.A., Cornish-Bowden, A., Groen, A.K., Heinrich, R.,
Kacser, H., Porteous, J.W., Rapoport, S.M., Rapoport, T.A.,
Stucki, J., Tager, J.M., Wanders, R.J.A. & Westerhoff, H.V.
(1985) Control analysis of metabolic systems. Trends Biochem. Sci.
10,16.
26. Thieringer, H.A., Jones, P.G. & Inouye, M. (1998) Cold shock
adaptation. Bioessays 20, 49–57.
27. Weber, M.H. & Marahiel, M.A. (2002) Coping with the cold: the
cold shock response in the Gram-positive soil bacterium Bacillus
subtilis. Phil. Trans. R. Soc. Lond. B 357, 895–907.
28. Aguilar, P.S., Cronan, J.E. & de Mendoza, D.
8
(1998) A Bacillus
subtilis gene induced by cold shock encodes a membrane phos-
pholipid desaturase. J. Bacteriol. 180, 2194–2200.
29. Klein,W.,Weber,M.H.W.&Marahiel,M.A.(1999)Coldshock
response of Bacillus subtilis: isoleucine-dependent switch in the
fatty acid branching pattern for membrane adaptation to low
temperature. J. Bacteriol. 181, 5341–5349.
30. Cronan, J.E. & Rock, C.O. (1996) Biosynthesis of membrane
lipids. In Escherichia coli and Salmonella Typhimurium: Cellular
and Molecular Biology (Neidhardt, F.C., ed.), pp. 612–636. ASM
Press, Washington DC.
31. Ganong, B.R. & Raetz, C.R. (1982) Massive accumulation of
phosphatidic acid in conditionally lethal CDP-diglyceride syn-
thetase mutants and cytidine auxotrophs of Escherichia coli.
J. Biol. Chem. 257, 389–394.
32. Switzer, R.L., Zalkin, H. & Saxild, H.H. (2002) Purine, pyri-
midine, and pyridine nucleotide metabolism. In Bacillus subtilis
and its Closest Relatives: from Genes to Cells (Sonnenshein, A.L.,
Hoch,J.A.&Losick,R.,eds),pp.255–269.ASMPress,Wash-
ington DC.
33. Gasson, M.J. (1983) Plasmid complements of Streptococcus lactis
NCDO 712 and other lactic streptococci after protoplast-induced
curing. J. Bacteriol. 154, 1–9.
34. Martinussen, J. & Hammer, K. (1995) Powerful methods to
establish chromosomal markers in Lactococcus lactis:ananalysis
of pyrimidine salvage pathway mutants obtained by positive
selections. Microbiology 141, 1883–1890.
Ó FEBS 2004 pyrG controls [CTP] and [dCTP] in L. lactis (Eur. J. Biochem. 271) 2445
. Expression of the
pyrG
gene determines the pool sizes of CTP
and dCTP in
Lactococcus lactis
Casper M. Jørgensen, Karin Hammer, Peter R. Jensen and. the pool sizes of CTP,
UTP and dCTP. In order to calculate how important the
Fig. 5. Effects of changing pyrG expression on the concentration of
dCTP. The
Ngày đăng: 16/03/2014, 16:20
Xem thêm: Báo cáo khoa học: Expression of the pyrG gene determines the pool sizes of CTP and dCTP in Lactococcus lactis doc, Báo cáo khoa học: Expression of the pyrG gene determines the pool sizes of CTP and dCTP in Lactococcus lactis doc