Microbiology (2011), 157, 1955–1967
DOI 10.1099/mic.0.047316-0
Overlap of replication rounds disturbs the
progression of replicating forks in a ribonucleotide
reductase mutant of Escherichia coli
Israel Salguero,1 Elena López Acedo2 and Elena C. Guzmán2
1
Correspondence
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
Elena C. Guzmán
[email protected]
2
Departmento de Bioquı́mica Biologı́a Molecular y Genética, Universidad de Extremadura,
06071 Badajoz, Spain
Received 1 December 2010
Revised
2 March 2011
Accepted 25 April 2011
Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of
deoxyribonucleotides (dNTPs). Surprisingly, Escherichia coli cells carrying the nrdA101 allele,
which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 6C
under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at
42 6C and that its degradation under restrictive conditions is prevented by the presence of
rifampicin. Nevertheless, the mere stability of the RNR protein at 42 6C cannot explain the
completion of chromosomal DNA replication in the nrdA101 mutant. We found that inactivation of
the DnaA protein by using several dnaAts alleles allows complete chromosome replication in the
absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed
in the nrdA101 mutant at 42 6C. As both inactivation of the DnaA protein and inhibition of RNA
synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of
forks per chromosome could be related to those effects. In support of this notion, we found that
avoiding multifork replication rounds by the presence of moderate extra copies of datA sequence
increases the relative amount of DNA synthesis of the nrdA101 mutant at 42 6C. We propose
that a lower replication fork density results in an improvement of the progression of DNA
replication, allowing replication of the entire chromosome at the restrictive temperature. The
mechanism related to this effect is also discussed.
INTRODUCTION
Ribonucleotide reductase (RNR) is the only enzyme
specifically required for the formation, under aerobic
conditions, of deoxyribonucleotides (dNTPs), the substrates of DNA synthesis in Escherichia coli (Nordlund &
Reichard, 2006). The active enzyme is a 1 : 1 complex of
two non-identical subunits named proteins R1 and R2,
each consisting of two nearly identical polypeptide chains
encoded by the genes nrdA and nrdB, respectively (Hanke
& Fuchs, 1983). The genes encoding the two RNR subunits
consititute an operon with poorly understood transcriptional control. Proteins including Fis, IciA, NrdR and
DnaA bind to specific sites in the promoter region to
regulate nrdAB expression, whereas several cis-acting sites
and an AT-rich region are important in coupling the gene’s
transcription to the cell cycle (Jacobson & Fuchs, 1998;
Han et al., 1998; Torrents et al., 2007). The best-known
defective RNR mutant in E. coli contains a thermolabile R1
subunit, encoded by the nrdA101 allele. This allele carries a
Abbreviation: RNR, ribonucleotide reductase.
Three supplementary figures are available with the online version of this
paper.
047316 G 2011 SGM
missense mutation, causing a change in amino acid 89
(L89P) (Odsbu et al., 2009). This leucine-to-proline
substitution is close to the ATP cone domain that is
located in the N-terminal region of the R1 protein and is,
according to the holoenzyme model, located close to the
R1–R2 interaction surface (Uhlin & Eklund, 1994),
although no structural analysis has been performed in the
mutant protein.
The RNR101 protein is inactivated in vitro after 2 min at
42 uC (Fuchs et al., 1972), although a thermoresistant
period of 50 min has been observed in vivo in the nrdA101
mutant strain (Guzmán et al., 2002). In light of the
observed mutant phenotype, RNR has been proposed to be
a component of the replication hyperstructure (Guzmán
et al., 2002, 2003; Molina & Skarstad, 2004; Guarino et al.,
2007a, b; Riola et al., 2007; Sánchez-Romero et al., 2010).
The presence of RNR as a structural element of this
hyperstructure would facilitate the provision of the four
dNTPs at the rate required for replication (Mathews,
1993). In addition, Riola et al. (2007) reported that the
nrdA101 mutant strain shows aberrant chromosome
segregation and defective cell division when incubated at
42 uC. This phenotype is not observed after inactivation of
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1955
I. Salguero, E. López Acedo and E. C. Guzmán
RNR by hydroxyurea, which suggests a relationship
between RNR and the chromosomal segregation machinery
through the maintenance of the replication hyperstructure
at the replication forks (Riola et al., 2007). A study by
Odsbu et al. (2009) supported these results, showing that
the discrete SeqA foci observed in an nrdAts mutant at the
permissive temperature were lost when incubated at 42 uC.
The thermoresistant period of the thermosensitive nrdA101
strain is extended when RNA synthesis is inhibited,
allowing cells to replicate complete chromosomes at
42 uC (Guzmán et al., 2002). This phenotype is surprising
because at 42 uC, the dNTP pool can only sustain a few
minutes of DNA replication and, as mentioned above, the
RNR encoded by nrdA101 is inactivated within 2 min in
vitro (Fuchs et al., 1972). Completion of chromosomal
replication at 42 uC in this thermosensitive mutant is not
specifically related to the inhibition of RNA synthesis
because we have verified that the runout replication at
42 uC was similar in the presence of rifampicin and in the
presence of chloramphenicol (Guzmán et al., 2002).
Consequently, an nrdA101 mutant is able to replicate the
entire chromosome at 42 uC when RNA synthesis is
prevented and can also perform thermoresistant replication
in the absence of protein synthesis, regardless of transcription inhibition.
In the present study, we explored the molecular mechanisms of the extensive thermoresistance of DNA replication
after the addition of rifampicin. We investigated three
possibilities that could explain this phenotype in the
presence of rifampicin. First, inhibition of the synthesis of a
certain negative-acting protein could benefit RNR101
stability. Second, the activity of the anaerobic class Ib
RNR encoded by the nrdHIEF operon could be induced
under restrictive conditions even though ordinarily it is
very poorly expressed and cannot take the place of the class
Ia RNR (Jordan et al., 1996). Finally, RNA and protein
syntheses are required to initiate chromosomal replication;
therefore, the inhibition of new initiation events could be
related to the extensive thermoresistant period. Inhibition
of DNA initiation can be achieved by inactivation of the
DnaA protein because it is absolutely required for the
initiation of chromosomal replication (reviewed by
Kaguni, 2006). Based on this behaviour, we studied the
thermoresistance of DNA replication in a double nrdA101
dnaA46ts mutant, which cannot initiate new rounds of
replication when incubated at 42 uC.
We show that RNR101 is stabilized in the presence of
rifampicin at 42 uC. Moreover, RNR101 stabilization at
42 uC is not sufficient to explain the completion of DNA
replication in the nrdA101 mutant strain under restrictive
conditions. We show that inactivation of the DnaA protein
allows complete chromosomal replication at 42 uC and
reverses the nucleoid and cell division defects in an nrdA101
mutant. These results point to the inhibition of new
initiation events provoked either by the presence of
rifampicin or by DnaA inactivation as the cause for the
1956
completion of chromosomal replication observed in
nrdA101 cells at 42 uC. In addition, the presence of moderate
extra copies of the datA sequence increases the relative
amount of DNA synthesis of the nrdA101 mutant at 42 uC.
This effect could be associated with the low number of
replication forks moving along the chromosome caused by
the extra copies of datA. We propose that a reduction in the
number of replication forks moving along the chromosome
would improve DNA replication progression.
METHODS
Bacterial strains and growth conditions. E. coli JS1018 (nrdA101
thyA arg his thi malA rpsL su xyl mtl) is a Pol+ Thy– lowrequirement derivative from the E1011 strain obtained from R.
McMacken (Stanford University, Stanford, CA, USA). All strains
used in this work were JS1018 derivatives. Strains were constructed
by the standard P1 transduction method. The JK607 strain was
constructed using the nrdA+ yfaL : : Tn5 strain, obtained from the
Coli Genetic Stock Center (Yale University New Haven, CT, USA),
as donor. Strain nrdA101 dnaA46 tna : : Tn10 was constructed using
the JRW27 strain (thr leu thi dnaA46 tnaA : : Tn10), obtained from
Dr J. Walker (University of Texas, Austin, TX, USA), as donor,
selecting for tetracycline (10 mg ml21) resistance and thermosensitivity at 42 uC and verifying the presence of the dnaA46 allele by
sequencing. The dnaA5 tnaA : : Tn10 and dnaA508 tnaA : : Tn10
alleles were derived from strains SS1750 and JC12390, respectively,
obtained from Dr S. Sandler (University of Massachusetts, Amherst,
MA, USA). The nrdE756 : : km and the lon725 : : km alleles were
derived from strains JW2650 and JW0429, respectively, obtained
from the Coli Genetic Stock Center. Plasmid pMOR6 containing
extra copies of datA sequence was obtained from K. Skarstad
(Morigen et al., 2001, 2003).
Bacteria were grown by shaking at 30 uC in M9 minimal medium
(MM9) containing M9 salts, 2 mg thiamine ml21, 0.4 % glucose,
20 mg ml21 of the required amino acids, 5 mg thymidine ml21 and
0.2 % Casamino acids, after a 1 : 200 dilution of an overnight culture.
Growth was monitored by measuring optical density at 550 nm.
Incubation at 42 uC or the addition of rifampicin (150 mg ml21) was
initiated at an OD550 of 0.1.
DNA synthesis measurements. DNA synthesis was determined by
growing the cells in MM9 medium containing 1 mCi ml21
(3.76107 Bq ml21) [methyl-3H]thymidine (20 Ci ml21) (ICN) and
assaying the radioactive acid-insoluble material.
Marker frequency analysis. Marker frequency analysis was
performed as described by Eliasson et al. (1996). Briefly,
chromosomal DNA was prepared from exponentially growing
cultures in MM9 medium at an OD550 of about 0.1. Growth was
immediately stopped by the addition of NaN3 to a final
concentration of 0.1 M, followed by storage on ice. As a control
for fully replicated chromosomes, we used DNA isolated from each
strain after 3 h of rifampicin treatment. Chromosomal DNA was
digested with EcoRI and HindIII overnight, and the fragments were
separated on 22 cm 1 % agarose gels at 35 V for 24 h. The fragments
were blotted onto nitrocellulose membranes by capillary transfer.
33
P-labelled probes were mixed together and hybridized to the
filters, and the intensity of each band was quantified by using a
PhosphorImager (Molecular Dynamics). The intensity of each band
relative to the intensity of the same band in the fully replicated
control was plotted as a function of the position on the E. coli
chromosome. Probes utilized were from sequences located at 24.2,
33.9, 47.8, 56.7, 66.5, 75.6, 78.7, 79, 85.6, 89.4 and 95.5 min on the
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Microbiology 157
Replication fork progression in an nrdA mutant
E. coli map and were made by PCR using primers commercially
obtained from Genosys. Labelled probes were created by random
primer reactions using [33P]dATP (NEN).
Flow cytometry. DNA content per cell was measured by flow
cytometry using a Bryte HS (Bio-Rad) flow cytometer, as described by
Skarstad et al. (1985) and Grigorian et al. (2003). When the cultures
reached an OD550 of 0.1, a portion was transferred into another flask,
and rifampicin (150 mg ml21) and cephalexin (50 mg ml21) were
added to inhibit new rounds of chromosome replication and cell
division, respectively. These treated cultures were grown for an
additional 4 h with continuous shaking, after which or at different
time intervals 400 ml of each culture was added to 7 ml 74 % ethanol.
Approximately 1.5 ml of each fixed sample was centrifuged, and the
pellets were washed in 1 ml ice-cold staining buffer (10 mM Tris,
10 mM MgCl2, pH 7.4, in sterile distilled water), and resuspended in
65 ml of staining buffer and 65 ml of staining solution (40 mg ethidium
bromide ml21 and 200 mg mithramycin A ml21). Cells were
incubated on ice in the dark for at least 30 min and analysed on
the Bryte-HS flow cytometer at 390–440 nm.
Microscopy. Phase-contrast and fluorescence micrographs were
obtained by treating the cultures with 100 mg chloramphenicol ml21
for 15 min to enhance visualization of nucleoids before fixing with
ethanol and staining with DAPI (49,6-diamidino-2-phenylindole).
Cells were fixed as for flow cytometry. To stain cells with DAPI,
400 ml of each fixed sample was centrifuged, washed with M9
salts and resuspended in 20 ml M9 salts. Ten microlitres was
then separately spread onto a flat surface of 0.5 mg DAPI ml21
in 1 % agar and 0.9 % NaCl on a microscope slide and dried at
42 uC for approximately 5 min in the dark before placing a
coverslip on the surface. Cells were visualized by phase-contrast
and fluorescence microscopy. A Nikon Eclipse E600 microscope
with a Hamamatsu C4742-95 camera was used to capture the
micrographs.
Western blot analysis. Quantification of the RNR subunit R1 was
performed by Western blot analysis. Cell lysates were obtained from
cultures of the different strains under the desired conditions. Equal
amounts of total protein were separated by electrophoresis on 8 %
polyacrylamide gels (SDS-PAGE). Proteins were transferred from the
acrylamide gels onto nitrocellulose membranes (Hybond-ELC;
Amersham). Subsequently, R1 protein was identified with a specific
polyclonal antibody, which was kindly provided by Dr J. Stubbe
(MIT, Cambridge, MA, USA). As a loading control, b-galactosidase
protein was detected with an anti-b-galactosidase antibody provided
by Acris Antibodies. Detection of these antibodies was performed by
using a horseradish peroxidase-conjugated IgG secondary antibody
(ImmunoPure Antibody; Pierce) and the Super Signal West Pico kit
(Pierce). Images were taken with the Molecular Imager FX system
(Bio-Rad).
RESULTS
RNR encoded by the nrdA101 allele is degraded at
42 ‡C
We previously reported that when the thermosensitive
nrdA101 mutant strain is incubated at 42 uC, it replicates
DNA for about 50 min, yielding stochastically arrested forks
before reaching the terminus and causing problems for
subsequent DNA segregation and cell division. However,
this strain is able to complete chromosome replication after
the addition of rifampicin or chloramphenicol at the
restrictive temperature (Guzmán et al., 2002). As gene
expression requires RNA and protein synthesis, one
explanation for this unexpected observation could be that
inhibition of the synthesis of some negative-acting product
could maintain the stability of RNR101 at 42 uC.
To explore this possibility, we analysed the stability of the
R1 subunit in the nrdA101 strain at 42 uC, in the absence
and presence of rifampicin, by Western blotting. Fig. 1(a)
reveals a decrease in amounts of R1 at the restrictive
temperature in the nrdA101 mutant. This effect was not
observed in the wild-type RNR, indicating a selective
Fig. 1. Western blot analysis of the R1 subunit
(NrdA) in the nrdA101 strain after different
times of incubation at 42 6C, after 180 min at
42 6C in the presence of rifampicin and in the
nrdA+ strain after 180 min at 42 6C (a), and in
the nrdA101 lon double mutant strain after
different times of incubation at 42 6C (b).
b-Galactosidase levels were used as a loading
control.
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I. Salguero, E. López Acedo and E. C. Guzmán
degradation of the protein encoded by the nrdA101 allele.
The degradation of RNR101 was dependent on RNA
synthesis as it was prevented by the addition of rifampicin
at the time of the temperature shift.
The anerobic class Ib RNR is encoded by the nrdHIEF
operon, and it is not essential for growth. It is ordinarily
very poorly expressed and cannot take the place of the class
Ia RNR (Jordan et al., 1996). Under standard laboratory
conditions, transcription of nrdAB is much greater than
that of nrdEF, but expression of both is elevated by DNA
damage or by DNA synthesis inhibitors (Gibert et al., 1990;
Jordan et al., 1996; Monje-Casas et al., 2001). Regarding
these observations, the supply of dNTPs synthesized by a
putative induced Ib RNR could be an alternative option to
explain the extended thermoresistant period of DNA
replication in the nrdA101 mutant under restrictive
conditions. We constructed the double nrdA101 DnrdE
mutant to evaluate DNA synthesis after the shift to 42 uC
in the presence or absence of rifampicin in the absence of
Ib RNR. The data in Table 1 show that the deficiency of
class Ib RNR does not result in the inhibition of DNA
synthesis in the nrdA101 background under any restrictive
conditions. These results indicate that both the DNA
synthesis at 42 uC and the completion of the DNA
replication rounds after rifampicin addition at 42 uC in
the nrdA101 mutant were not due to the activity of the
putatively induced Ib RNR.
Heat-shock proteases are known to be induced after being
shifted to 42 uC. Among them, the Lon protease plays a
major role in protein quality control. A high number of
proteins involved in functions from housekeeping to
regulation have been identified as Lon substrates
(Gottesman et al., 1997; Gottesman, 2003; Tsilibaris et al.,
2006). We analysed the amount of the R1 subunit in the
double mutant strain nrdA101 lon at 42 uC. We found
considerable stabilization of RNR101 in the absence of Lon
(Fig. 1b). Nevertheless, the nrdA101 lon mutant only
slightly increased the amount of DNA synthesis at 42 uC
compared with the nrdA101 mutant, in contrast to the
complete chromosome replication observed when RNA
synthesis was inhibited (Table 1; see also Supplementary
Fig. S1, available with the online version of this paper).
Overall, the phenotype of the double nrdA101 lon mutant is
quite similar to that of the single nrdA101 mutant,
indicating that the stabilization of RNR101 after rifampicin
addition is not related to the completion of chromosomal
replication at 42 uC in the presence of the drug.
The presence of dnaAts alleles allows the
nrdA101 mutant to complete chromosomal
replication at 42 ‡C
RNR coded by the nrdEF alleles is not responsible
for the DNA synthesis in the nrdA101 mutant at
42 ‡C
Because synthesis of both RNA and protein is required
for new chromosome initiation events, we speculated
that the extended thermoresistant period of chromosome
Aerobic growth of E. coli depends on a functional class Ia
RNR encoded by the nrdAB operon (Jordan et al., 1996).
Table 1. Cell cycle parameters and ori/ter ratio of nrdA101 and dnaA derivatives growing at 30 6C and after 4 h of incubation at
42 6C in the presence or absence of rifampicin
The percentage increase in the amount of DNA (DG) and the subsequent parameters, n and ori/ter, were obtained from quantitative analysis of the
results shown in Figs 2, 4, 7 and 8.
Strain
t*
DG 30 6CD
nd
ori/ter (2n)
42 6C§
42 6C/
DG 30 6C||
+
nrdA
nrdA101
nrdA101 lon
nrdA101 nrdE
dnaA46
nrdA101 dnaA46
nrdA101/pMOR6
78
79
90
80
80
80
81
55
103
95
100
47
45
73
1.37
2.36
2.2
2.30
1.19
1.15
1.76
2.58
5.13
4.60
4.92
2.28
2.21
3.38
+Rif
”Rif
57
105
95
90
43
46
72
2
48
55
50
47
42
70
2
0.46
0.51
0.50
1.01"
0.91"
0.95
*Generation time (min).
DIncrease (%) in the amount of DNA 4 h after addition of rifampicin at 30 uC to inhibit new initiation events, while ongoing forks are allowed to
finish (Pritchard & Zaritsky, 1970).
dNumber of replication cycles per chromosome obtained from DG values at 30 uC (Sueoka & Yoshikawa, 1965; Jiménez-Sánchez & Guzmán, 1988).
§Increase (%) in the amount of DNA after 4 h of incubation at 42 uC.
||Increase (%) in the amount of DNA after 4 h of incubation at 42 uC in the absence of rifampicin relative to the value of D G 30 uC.
"Note that in the dnaA46 mutants new chromosomal initiation is inhibited at 42 uC in either the absence or the presence of rifampicin and the
values obtained are the highest that could be expected.
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Microbiology 157
Replication fork progression in an nrdA mutant
replication in the nrdA101 mutant at 42 uC in the absence
of RNA or protein synthesis could be caused by the
inhibition of new DNA initiation. To test this hypothesis,
we constructed a double nrdA101 dnaA46 mutant strain.
The DnaA protein is absolutely required for initiation of
chromosomal replication, and the dnaA46 allele encodes a
thermolabile DnaA46 protein that inhibits new initiation
events at 42 uC (reviewed by Kaguni, 2006), whereas RNA
and protein synthesis remain active. DNA synthesis by the
nrdA101 dnaA46 strain at 42 uC was quantified in the
absence or presence of rifampicin. After new initiations of
replication were inhibited by inactivation of DnaA46 at
42 uC, there was a relative increase in DNA synthesis and a
thermoresistant period similar to that obtained after
incubation at 42 uC in the presence of rifampicin (Fig.
2d, Table 1). Flow cytometry revealed that in either the
presence or the absence of rifampicin, the double nrdA101
dnaA46 mutant was able to replicate complete chromosomes at the restrictive temperature (Fig. 2d), in contrast
to the single nrdA101 mutant, which failed to complete
replication rounds at the restrictive temperature in the
absence of rifampicin (Fig. 2b). The flow cytometry profile
of the nrdA101 dnaA46 strain at 42 uC showed two, three
and four chromosomes per cell, indicating an asynchronous replication phenotype that was also observed in the
nrdA+ dnaA46 strain incubated at 42 uC in the presence or
absence of rifampicin (Fig. 2c). This phenotype is explained
solely by the presence of the dnaA46 allele, as previously
reported (Skarstad et al., 1988).
To verify further the completion of chromosomal replication at 42 uC in the nrdA101 dnaA46 strain, marker
Fig. 2. Runout DNA synthesis (left panels) and flow cytometry profiles (right panels) after 4 h of treatment at 42 6C in the
presence of cephalexin or cephalexin plus rifampicin of the (a) nrdA+ dnaA+, (b) nrdA101 dnaA+, (c) nrdA+ dnaA46 and (d)
nrdA101 dnaA46 strains.
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I. Salguero, E. López Acedo and E. C. Guzmán
frequency analysis was performed under a variety of
conditions. As previously reported (Guzmán et al., 2002),
incubation of the nrdA101 single mutant for 4 h at the
restrictive temperature (Fig. 3c) resulted in a marker
frequency distribution similar to that obtained with an
exponentially growing culture at 30 uC (Fig. 3a). In
contrast, 4 h after being shifted to 42 uC, the nrdA101
dnaA46 strain showed a flat distribution of marker
frequencies (Fig. 3d), which indicates the completion of
ongoing chromosomal replications, as observed after
addition of rifampicin at 42 uC, in either nrdA101 or
nrdA101 dnaA46 mutants (Fig. 3e, f). These results
demonstrate that the nrdA101 dnaA46 strain is able to
execute complete chromosomal replication at the restrictive temperature.
It should be noted that there was a lower ori/ter ratio in the
double nrdA101 dnaA46 mutant strain than in the nrdA101
parental strain (Fig. 3a, b). To confirm this effect, runout
DNA synthesis (DG) and flow cytometry at 30 uC after the
addition of rifampicin and cephalexin were performed in
strains nrdA+, nrdA101, nrdA+ dnaA46 and nrdA101
dnaA46 (Fig. 4). The DG value is defined as the relative
runout DNA synthesized after inhibiting new initiation
events, while ongoing forks are allowed to finish (Pritchard
& Zaritsky, 1970). From the experimental DG value, n can be
calculated from the algorithm DG5[2nn ln2/(2n21)]21
(Sueoka & Yoshikawa, 1965). Thus, the relative amount of
DNA synthesized after inhibition of initiation of replication
depends only on the number of replication cycles per
chromosome before the inhibition; the value of n was
obtained by using a software (Replicon) developed in our
laboratory (Jiménez-Sánchez & Guzmán, 1988). The
calculated values of ori/ter, 2n, are shown in Table 1. Fig. 4
shows that the presence of the dnaA46 allele reduced the ori/
ter ratio in the nrdA101 mutant at 30 uC, explaining the
decrease in the runout value (DG) obtained at 42 uC with or
without rifampicin in the nrdA101 dnaA46 mutant (Fig. 2d,
Table 1). This effect has been previously described by other
groups using dnaA defective alleles alone (Gon et al., 2006)
or combined with dnaX alleles that impaired the progression
of DNA replication (Skovgaard & Løbner-Olesen, 2005).
The presence of dnaAts alleles suppresses the
segregation and division defects of an nrdA101
mutant
Growth of an nrdA101 strain at the restrictive temperature
causes filamentation and affects the DNA distribution
Fig. 3. Marker frequency relative to the replication terminus of the
nrdA101 (left panels) and nrdA101 dnaA46 (right panels) strains
growing in minimal medium at 30 6C (a, b), 4 h after the shift to
42 6C (c, d) or after the temperature shift in the presence of
rifampicin (e, f).
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Fig. 4. Runout DNA synthesis and flow cytometry profiles (insets)
after 4 h of treatment in the nrdA+ dnaA+(a), nrdA101 dnaA+(b),
nrdA+ dnaA46 (c) and dnaA46 nrdA101 (d) strains after the
addition of rifampicin and cephalexin at 30 6C.
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Microbiology 157
Replication fork progression in an nrdA mutant
throughout the cell (Guzmán et al., 2003; Riola et al., 2007;
Odsbu et al., 2009). Phase-contrast microscopy of the
double nrdA101 dnaA46 mutant strain growing at the
restrictive temperature showed no filamentation (Fig. 5).
Nucleoid segregation was studied by fluorescence microscopy of DAPI-stained cells after growth for 4 h at the
restrictive temperature in the presence of cephalexin (to
inhibit cell division). Micrographs show correct nucleoid
segregation in the double nrdA101 dnaA46 mutant, in
contrast to the abnormal DNA segregation observed in the
nrdA101 strain (Fig. 5). The aberrant division and nucleoid
segregation pattern observed in the nrdA101 strain have
been attributed to the breakdown of coupling between
replication and cell division (Dix & Helmstetter, 1973;
Riola et al., 2007). Our results are consistent with this
notion; they show that cell filamentation and alteration of
nucleoid segregation observed in the nrdA101 strain at
42 uC were eliminated when complete chromosomal
replication was allowed through the introduction of the
dnaA46 allele.
To verify that the phenotype observed in the nrdA101
dnaA46 mutant is not specific to the dnaA46 allele, we
constructed nrdA101 dnaA5 and nrdA101 dnaA508 double
mutant strains. Measurements of DNA synthesis in these
strains after incubation at 42 uC were similar to those
observed for the dnaA46 strain (data not shown), and the
effects on cell division and nucleoid segregation caused by
the nrdA101 mutation were reversed by the presence of any
of these two dnaA alleles (Fig. 5).
To further study these effects on cell division and DNA
segregation after shifting to a non-permissive temperature,
a time-course experiment was performed. OD550 values,
cell mass, DNA content per cell, cell division and nucleoid
segregation were analysed in the single nrdA101 and
dnaA46 mutants as well as in the double nrdA101
dnaA46 mutant at 1, 2, 3, 4 and 6 h after the shift to
42 uC (Fig. 6, Supplementary Fig. S2). The results indicated
that after the temperature shift the nrdA101 dnaA46 strain
maintained protein synthesis for two generations (measured by OD550); cell division was allowed, as the relative
number of cells increased by a factor of three (measured as
the number of cells per volume unit); cell mass was
maintained; and the DNA content per cell decreased
slightly (Fig. 6). Similar results were found in the single
dnaA46 mutant. In contrast, cell division was inhibited
while protein synthesis continued in the nrdA101 mutant;
cell mass consequently increased and DNA content per cell
was maintained, as previously described (Guzmán et al.,
2002; Riola et al., 2007). It should be noted that the nrdA+
dnaA46 and nrdA101 dnaA46 strains continued accumulating biomass at 42 uC for two generations but not
indefinitely, as has been observed in other dnaA46 genetic
backgrounds. These results explain why cell filamentation
was not observed in the dnaA46 mutant strains in either
Fig. 5. Phase-contrast microscopy of cells of the nrdA101, nrdA101 dnaA46, nrdA101 dnaA5, nrdA101 dnaA508, nrdA+ and
nrdA+ dnaA46 strains growing at 30 6C (upper panels) or 4 h after the shift to 42 6C (middle panels), and fluorescence
microscopy of DAPI-stained cells 4 h after the shift to 42 6C in the presence of cephalexin (lower panels). Bar, 10 mm.
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I. Salguero, E. López Acedo and E. C. Guzmán
Fig. 6. Time-course quantification of biomass,
DNA content per cell, mass per cell and number
of cells per volume of the nrdA+ dnaA+(a),
nrdA101 dnaA+ (b), nrdA+ dnaA46 (c) and
nrdA101 dnaA46 (d) strains after the shift to
42 6C in the absence of cephalexin. The bars in
the graphs represent (from left to right) relative
values of: light scattering (mass per cell), OD550
(biomass), OD550¾(1/light scattering) (number
of cells/volume), and fluorescence (DNA content per cell).
the presence or the absence of the nrdA101 allele (Fig. 5,
Supplementary Fig. S2). DNA segregation became gradually aberrant in the nrdA101 mutant after the shift to 42 uC,
while this alteration was suppressed in the nrdA101 dnaA46
double mutant (Supplementary Fig. S2).
All these results suggest that the inhibition of new DNA
initiation events is a physiological condition that allows the
thermosensitive RNR101 to complete chromosome replication, suppressing the DNA segregation and cell division
defects observed in the nrdA101 mutant at the restrictive
temperature.
Low overlap of replication rounds improves
progression of elongating forks in the nrdA101
mutant under restrictive conditions
The fact that the inhibition of DNA initiation increases the
thermoresistant period in an nrdA101 mutant could be
explained by a lower replication fork density on the
chromosome as a consequence of the inhibition of new
initiation events. The low density of forks could improve
the progression of the ongoing replication forks, maintaining the activity of fork-associated RNR101 until the
end of chromosomal replication.
To investigate this hypothesis we determined DNA
synthesis at 42 uC in the nrdA101 strain harbouring extra
copies of the datA sequence. There are 308 DnaA boxes
[TTAT(C/A)CA(C/A)A] in the E. coli chromosome that
have variable affinity to DnaA (Schaper & Messer, 1995).
Among these DnaA boxes, a strong binding region, datA
(DnaA titration), which carries five boxes, has been
1962
identified (Kitagawa et al., 1996). The datA site titrates
unusually large amounts of DnaA protein in vivo (Kitagawa
et al., 1996). It has been shown that moderate amplification
of datA decreases the ori/ter ratio relative to that of the
wild-type and consequently the overlap of the replication
rounds (Morigen et al., 2001, 2003). Taking advantage of
this feature, we constructed an nrdA101 strain harbouring
the pMOR6 plasmid, a derivative of the moderate-copynumber plasmid pACYC177 that carries the chromosomal
datA locus (Morigen et al., 2001).
The presence of moderate numbers of extra copies of the
datA sequence in the nrdA101/pMOR6 strain yielded a
lower runout synthesis (DG) after the addition of
rifampicin at 30 uC than the nrdA101 strain without the
plasmid, reducing the overlap of replication rounds in the
nrdA101 background from 2.36 to 1.76 (Fig. 7a, Table 1).
After 4 h of incubation at 42 uC we found a similar amount
of DNA synthesis, in either the presence or the absence of
rifampicin under restrictive conditions. This result differs
from that obtained in the nrdA101 mutant, whose DNA
synthesis at 42 uC is half that observed at 42 uC when new
initiations were inhibited (Fig. 2b, Table 1). Given that in
the nrdA101/pMOR6 strain DNA initiation is not inhibited
by the shift to 42 uC, fully replicated chromosomes at 42 uC
are not detected (Fig. 7b), although the amount of runout
DNA synthesis at 42 uC was similar in the presence or in
the absence of the drug.
These results support the hypothesis of a decrease in
replication fork density as an important factor to improve
the progression of DNA replication in the nrdA101 mutant
at the restrictive condition.
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Replication fork progression in an nrdA mutant
Fig. 8. Runout DNA synthesis of the nrdA101 strain after the shift
from 30 to 42 6C in the absence ($) or in the presence (&) of
rifampicin, or in the presence of rifampicin and hydroxyurea (m); at
30 6C in the presence of rifampicin (#); or in the presence of
rifampicin and hydroxyurea (g).
DISCUSSION
Fig. 7. (a) Runout DNA synthesis of the nrdA101/pMOR6 strain
after the shift from 30 to 42 6C in the absence ($) or in the
presence (&) of rifampicin, or at 30 6C in the presence
of rifampicin (#). (b) Flow cytometry profile of the nrdA101/
pMOR6 strain after 4 h at 42 6C with cephalexin in the
absence (dashed line) or in the presence (solid line) of
rifampicin.
The free dNTP pool is not shared among different
replication forks
One possibility to explain how a decrease in the number of
replication forks could increase the time of active
elongating forks could be that blocking the emergence of
new replication forks may increase the amount of free
dNTP available per fork. We evaluated this possibility by
treating a culture of the nrdA101 strain with rifampicin
and/or hydroxyurea at 30 or 42 uC. The results in Fig. 8
show that, even though new initiations of replication were
inhibited by rifampicin, inactivation of RNR by hydroxyurea stopped DNA synthesis instantaneously at 30 and at
42 uC. This observation clearly differs from the DNA
synthesized by the nrdA101 mutant when incubated at
42 uC, and indicates that, if replication forks obtained the
required precursors from a free dNTP pool, this amount
must be very low as it permitted practically undetectable
DNA synthesis.
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In this work, we used three approaches to investigate the
physiological condition that maintains the DNA replication active in the nrdA101 mutant, yielding full replicated
chromosomes at 42 uC, when RNA or protein synthesis is
inhibited.
First, we have shown that the amount of the RNR subunit
R1 decreases gradually with incubation at the high
temperature and that this effect is prevented by rifampicin.
Nevertheless, the degradation of the R1 subunit in the
double nrdA101 lon mutant at 42 uC is greatly reduced
without a concomitant increase in the amount of DNA
synthesis. From these results we conclude that the mere in
vivo stabilization of RNR101 at 42 uC is not sufficient to
allow the completion of chromosomal replication in the
nrdA101 mutant under restrictive conditions.
On the other hand, we have demonstrated that the Ib RNR
encoded by the nrdEF operon, which is inactive under
standard laboratory conditions but can be induced under
stress conditions, is not responsible for the completion of
the DNA chromosomal replication observed in the
nrdA101 mutant at 42 uC in the presence or absence of
rifampicin.
Inhibition of new replication cycles allows entire
chromosomal replication in the nrdA101 mutant
under restrictive conditions
To examine whether the extension of the thermoresistant
period of RNR101 was a consequence of the absence of new
initiations of replication caused by the inhibition of RNA
or protein synthesis, we introduced the dnaA46 allele into
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I. Salguero, E. López Acedo and E. C. Guzmán
the nrdA101 strain. We found that the DNA synthesis
permitted at 42 uC was sufficient to replicate entire
chromosomes, regardless of protein synthesis inhibition.
Similar results were obtained by using the dnaA5 and
dnaA508 alleles, which encode thermolabile DnaA proteins
affected in different domains (Messer, 2002). According to
these results, the completion of chromosome replication at
42 uC in the nrdA101 strain could be ascribed to the
inhibition of further DNA initiations, either by inhibition
of protein synthesis or by thermal inactivation of the DnaA
protein.
Nevertheless, other possibilities could be considered. In
addition to its role in the initiation of chromosomal
replication, the DnaA protein acts as a transcriptional
regulator controlling the expression of several genes,
including dnaA, mioC, rpoH and the nrdAB operon
(reviewed by Messer & Weigel, 2003; Gon et al., 2006;
Herrick & Sclavi, 2007; Olliver et al., 2010). Furthermore,
the dnaA46 strain has been shown to increase expression of
the nrdAB operon at 42 uC and, to a lesser extent, at 30 uC
(Løbner-Olesen et al., 2008), a feature that has been
corroborated in the double nrdA101 dnaA46 mutant
(Supplementary Fig. S3). This situation could be related to
the extended thermoresistant period of DNA synthesis.
However, in the nrdA101 dnaA46 mutant strain, the
chromosome is fully replicated at 42 uC despite inhibition
of protein synthesis, a condition where overexpression of
RNR101 cannot take place. Besides, the mere presence of
RNR101 does not account for the complete chromosome
replication at 42 uC, as indicated by the double nrdA101 lon
mutant. Furthermore, overexpresion of the nrdAB operon
from a plasmid leading to a doubling of the enzyme activity,
as measured in cell-free extracts, causes the steady-state
pools of dATP, dCTP and dTTP to increase about twofold,
but no increase in dGTP (Wheeler et al., 2005). Although it
is not possible to ascertain the effective dNTP concentrations at the replication point resulting from overexpression
of RNR101, those results indicate that overexpresion of the
enzyme did not necessarily increase the actual supply of
dNTP used in DNA replication. Therefore, overexpression
of RNR101 after the temperature shift could be ruled out as
the cause for the completion of chromosomal replication at
42 uC in the nrdA101 dnaA46 mutant.
Reducing the number of replication forks
alleviates DNA replication progression in the
nrdA101 mutant
We studied the possibility that inhibition of new initiation
events could improve replication progression in the
nrdA101 mutant at the restrictive temperature by decreasing the number of replication forks along the chromosome.
We found that the presence of moderate numbers of extra
copies of the datA sequence on the plasmid pMOR6
reduces the overlap of the replication cycles from 2.36 to
1.76 (Table 1). This means that, at every new initiation, the
number of replication forks per chromosome in the
1964
nrdA101 mutant increases from 6 to 14. In contrast, in
the presence of extra copies of datA the number of forks
per chromosome increases from two to six. As predicted by
this hypothesis, we observed an increase in the relative
amount of DNA synthesis under restrictive conditions,
reaching the same level as after addition of rifampicin.
Exploring the mechanism linking the two processes, i.e. the
reduction of the overlap of replication rounds and the
capacity of the nrdA101 mutant to replicate the entire
chromosome, we considered several possibilities. The first
is that the inhibition of new DNA initiation events could
spare the demand for dNTPs of the cell, as fewer
replication forks will be active. This notion would imply
that there is a free dNTP pool that can be shared by the
functioning replicating forks. This is unlikely, as it is
known that bacterial cell pools of dNTPs are very low and
only allow a few minutes of a single replicating fork (Pato,
1979; Kornberg & Baker, 1991). In this regard, we found
that even though new initiations of replication were
inhibited by addition of rifampicin, inactivation of RNR
by hydroxyurea stopped DNA synthesis instantaneously at
30 or 42 uC. This indicates that the cytoplasmic dNTP pool
cannot maintain the replication even in a scenario where
the total demand for dNTPs is reduced, and consequently
RNR101 must be active at the restrictive temperature
during the whole period of DNA synthesis.
The second alternative would be that there is a weak
residual activity of RNR101 at non-permissive temperature, but that this is insufficient to feed multiple
replication forks unless the number of forks is reduced.
This possibility cannot be ruled out; however, it is difficult
to reconcile with several results. (1) According to this
hypothesis, one would expect a faster replication at 30 uC
than at 42 uC after inhibition of initiation. But we do not
observe any difference between DNA synthesis at 30 uC
after rifampicin addition, when RNR101 would be ‘fully
operative’, and at 42 uC in the presence of the drug, when
the enzyme would maintain only a residual activity. (2)
The total RNR101 residual activity at 42 uC should increase
under conditions where the stability of the enzyme is
higher, as in the nrdA101 lon double mutant; however, a
significant increase of DNA synthesis in the double mutant
at 42 uC was not observed.
Another mechanism to explain this phenotype could be
related to the chromosome structure. Strains harbouring
the nrdA101 allele overlap two and three replication
rounds. This situation generates a multifork chromosome
structure (6 and 14 forks, respectively) with a potential
negative effect on the progression of replication forks.
Reducing the overlap of replication rounds at 42 uC, either
by blocking new initiations or as a result of the effect of
extra datA copies, could therefore improve the defective
progression of replication forks in the nrdA101 mutant
(Guarino et al., 2007a). In this context, Odsbu et al. (2009)
have shown that replication forks grouped in discrete foci
and the number of forks per focus can increase if more
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Microbiology 157
Replication fork progression in an nrdA mutant
replication rounds coexist in the same cell. This observation suggests that forks from different replication cycles
can localize together inside the cell, facilitating the
possibility of interference between them.
The results presented here support the general concept of
a reduction in the number of replication forks as a critical
condition in the progression of defective replication forks
along the chromosome. This effect has been observed in
other genetic backgrounds, suggesting that the initiation
and elongation stages of replication are not independent
processes. It has been reported that deficient DnaA
protein reduces the number of replication forks per
chromosome, re-establishing the fork progression in
replisome mutants dnaX (Skovgaard & Løbner-Olesen,
2005; Walker et al., 2006) and suppressing the sensitivity
to UV irradiation, AZT (azidothymidine) or hydroxyurea
in a seqA mutant (Sutera & Lovett, 2006; Molt et al.,
2009). Moreover, it has been shown that cells carrying the
deletion of the DnaA-binding site R4 in the minimal oriC
sequence exhibit deficient chromosome initiation, suppressing the replication defects caused by hyper-initiation
in a DseqA strain (Stepankiw et al., 2009). In contrast,
overproduction of the DnaA protein decreases viability in
a strain deficient in recombinational repair (Grigorian
et al., 2003), probably due to overlapping replication
cycles; this yields a multi-forked structure near oriC that
decreases the replication rate around this region (Atlung
& Hansen, 1993). Finally, the extensive fork collision and
collapse caused by over-initiation of DNA replication in
dnaA(cos) mutants has been shown to be suppressed by
decreasing the efficiency of replication fork movement in
vivo with defective holC or ndk alleles (Nordman et al.,
2007). In the nrdA101 mutant, replication forks have been
shown to stall more frequently than in a wild-type strain
(Guarino et al., 2007a). This could result in collisions
between ongoing and stalled forks, which would be
prevented if new initiations were not allowed or if
multifork replication rounds were prevented.
Inhibition of initiation suppresses the
morphological defects of an nrdA101 mutant
In addition to the completion of chromosomal replication
after the inhibition of initiation in nrdA101 dnaA46 cells at
42 uC, we also observed suppression of the alterations in
DNA segregation and cell division observed in the nrdA101
mutant strain at the restrictive temperature (Riola et al.,
2007; Odsbu et al., 2009). These results suggest that the
morphological alterations observed in the nrdA101 mutant
at 42 uC could be related to the presence of stalled
replication forks. This effect has been described under
other conditions, including UV irradiation, thymine
starvation or mitomycin treatments and inversion of the
Ter sequences (Jaffé et al., 1986; Hill et al., 1997), and in
dnaN59ts and dnaG2903ts mutants, where it has also been
associated with stalled replication forks (Grompe et al.,
1991; Kawakami et al., 2001).
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To decipher the mechanisms involved in coupling cell
division and chromosomal replication, we can study
conditions where the link between these processes has
been disturbed. The results presented here suggest that the
overlap of replication rounds could be a significant element
in understanding the correlation between replication fork
progression, nucleoid segregation and cellular division as a
general physiological mechanism connecting these processes in E. coli.
ACKNOWLEDGEMENTS
We thank J. R. Walker, S. Sandler and K. Skarstad for strains and
plasmids and J. Stubbe for the kind gift of the anti-R1 antibody. We
are very grateful to Encarna Ferrera for her technical help and to
Carmen Mata Martı́n for her collaboration in the laboratory work.
We are indebted to Alfonso Jiménez-Sánchez and Estrella Guarino for
discussions about this work and a critical reading of the manuscript.
This work was supported by grants from the Junta de Extremadura
(PRI07A001) and Ministerio de Educación y Ciencia (BFU200763942). I. S. acknowledges a studentship from the Ministerio de
Educación y Cultura (FPU AP2002-1157).
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