Determination of concentration control coefficients The experimental data of reporter gene activity from the strains with altered pyrG expression and corresponding growth rate or concent
Trang 1Expression of the pyrG gene determines the pool sizes of CTP
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
pyrGexpression 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
1Synthesis of ribonucleotides and deoxyribonucleotides is
an essential part of cellular metabolism, as synthesis of
RNArequires ribonucleotides and DNAreplication 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
pyrGgene encoding CTP synthase (EC 6.4.3.2) in L lactis
and B subtilis, as pyrG expression is probably regulated
by an attenuation mechanism responding to the CTP
concentration in the cell [6,7] The reaction catalyzed by
CTP synthase (UTP + glutamine + A TP fi CTP +
glutamate + ADP + Pi) 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 TAcloning kit from Invitrogen Chromosomal DNApurified from MG1363 was used as a template for the PCR amplification
Growth medium and growth conditions
L lactisstrains were grown either in M17 broth supplied with 1% (w/v) glucose or in defined SAmedium [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)
Trang 21% (w/v) glucose at 30C 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)1in SAmedium and to
1000 lgÆmL)1in M17 medium
Transformation of DNA toL 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)1for phenotypic expression
Isolation of chromosomal DNA fromL lactis Chromosomal DNAwas isolated as described in [13] Isolation of strains with alteredpyrG expression APCR 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 XhoI and PstI and ligated in plasmid pLB86 also digested with XhoI and PstI 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]
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]
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 mRNAis 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 ribonucleoribonucleo-tide reductase.
Trang 3M17 plates with glucose, erythromycin, chloramphenicol,
cytidine at 1000 lgÆmL)1and
5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal) at 90 lgÆmL)1
Growth experiments
The strains were grown over night at 30C on SA plates
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 30C This growing culture was diluted and
used to inoculate 10 mL of SAmedium with glucose,
erythromycin and cytidine so that the next day the growth
experiment could start with an exponentially growing
culture (D436< 0.8)
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 SAmedium with glucose and erythromycin was
inoculated to D436of 0.025 and the growth monitored by
measuring D436 A t D436of 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 30C as previously described [14], except
that cell density was measured at 436 nm and the specific
activity was therefore determined as A420/D436per minute
per mL of culture (units/D436)
Nucleotide pool determinations
The concentration of nucleotides in the cell was determined
by thin layer chromatography on PEI-plates of 33PO43
-labeled nucleotides extracted from exponentially growing
cells at a cell density of D436¼ 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/D436
the functions f(z)¼ 2.49–2.49 · exp() 0.0237 · z1.362) 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)
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/D436 The
CTP concentration data were then fitted to the
func-tion f(x)¼ 3.81–3.81 · exp()0.00101 · x1.352), where x is
expression of the reporter genes relative to the wild-type
level of 9.5 units/D436 When the CTP concentration data
were fitted to the function f(x)¼ 0.0068 · 0.998x · x1.225,
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 · x and f(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 equation¼ 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 alteredpyrG expression Strains with different constitutive expression levels of the
6pyrGgene 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] Adegenerate primer con-taining a promoter with consensus)10 and )35 promoter regions separated by a randomized spacer of 17 nucleo-tides plus a 3¢-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 DNAfragment 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 only 20 or 50 lg of cytidine per milliliter [6,8] However,
Trang 4L lactis pyrGmutants 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 ofpyrG 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 N4-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/D436 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/D436 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
ofL 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/D436, 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 ofpyrG expression
Figure 4Ashows 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 Concentraconcentra-tions 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.
Trang 5size 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 pyrG 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.
Trang 6enzyme 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 C½CTPPyrG 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 to¼ 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 mRNAturnover
[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 15C and
30C 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 15C than at 30 C compared
to strains with normal pyrG expression One of these strains has 43% pyrG expression relative to the wild-type and no growth defect at 30C 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 30C compared to the wild-type, but these strains are growth impaired at 15C, as they do not grow, even after 8 days of incubation at 15C 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)1and 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 +++
Trang 7The 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 DNAsupercoiling 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 normal 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
pyrGexpression 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
30C, 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 vivoactivity 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 lactiscell 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 Avariation 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)
Astrain with pyrG expression reduced to 43% of the wild-type level has decreased CTP and dCTP pool sizes and show reduced growth at 15C, whereas no effect on growth was observed at 30C (Table 2) The growth defect is relieved by cytidine addition, suggesting that the observed slow growth at 15C 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 15C of strains with decreased CTP and dCTP pool sizes at 30C Reduced synthesis or activity of CTP synthase at 15C 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 the cell membrane to growth at 15C resulting in slow growth at this temperature The importance of CTP synthase in E coli phospholipid biosynthesis was investi-gated in a pyrG 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.
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